Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS

ABSTRACT

This invention relates to antiviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), particularly treatment regimens including a protease inhibitor. The invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays.

This application is a continuation in part of U.S. application Ser. No.09/591,899, filed Jun. 12, 2000, now U.S. Pat. No. 6,869,759, and isalso a continuation in part of PCT International Application No.PCT/US00/17178, filed Jun. 22, 2000, the contents of each of which arehereby incorporated by reference into this application.

Throughout this application, various references are referred to withinparenthesis. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

TECHNICAL FIELD

This invention relates to antiretroviral drug susceptibility andresistance tests to be used in identifying effective drug regimens forthe treatment of human immunodeficiency virus (HIV) infection andacquired immunodeficiency syndrome (AIDS). The invention further relatesto the means and methods of monitoring the clinical progression of HIVinfection and its response to antiretroviral therapy using phenotypic orgenotypic susceptibility assays. The invention also relates to novelvectors, host cells and compositions for carrying out phenotypicsusceptibility tests. The invention further relates to the use ofvarious genotypic methodologies to identify patients who do not respondto a particular antiretroviral drug regimen. This invention also relatesto the screening of candidate antiretroviral drugs for their capacity toinhibit viral replication, selected viral sequences and/or viralproteins. More particularly, this invention relates to the determinationof protease inhibitor (PRI) susceptibility using phenotypic or genotypicsusceptibility tests. This invention also relates to a means and methodfor accurately and reproducibly measuring viral replication fitness.

BACKGROUND OF THE INVENTION

HIV infection is characterized by high rates of viral turnoverthroughout the disease process, eventually leading to CD4 depletion anddisease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature343, 117–122 and Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature373, 123–126. The aim of antiretroviral therapy is to achievesubstantial and prolonged suppression of viral replication. Achievingsustained viral control is likely to involve the use of sequentialtherapies, generally each therapy comprising combinations of three ormore antiretroviral drugs. Choice of initial and subsequent therapyshould, therefore, be made on a rational basis, with knowledge ofresistance and cross-resistance patterns being vital to guiding thosedecisions. The primary rationale of combination therapy relates tosynergistic or additive activity to achieve greater inhibition of viralreplication. The tolerability of drug regimens will remain critical,however, as therapy will need to be maintained over many years.

In an untreated patient, some 10¹⁰ new viral particles are produced perday. Coupled with the failure of HIV reverse transcriptase (RT) tocorrect transcription errors by exonucleolytic proofreading, this highlevel of viral turnover results in 10⁴ to 10⁵ mutations per day at eachposition in the HIV genome. The result is the rapid establishment ofextensive genotypic variation. While some template positions or basepair substitutions may be more error prone (Mansky L M, Temin H M (1995)J Virol 69, 5087–5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al.(1994) Antimicrob Agents Chemother 38, 268–274), mathematical modelingsuggests that, at every possible single point, mutation may occur up to10,000 times per day in infected individuals.

For antiretroviral drug resistance to occur, the target enzyme must bemodified while preserving its function in the presence of the inhibitor.Point mutations leading to an amino acid substitution may result inchanges in shape, size or charge of the active site, substrate bindingsite or in positions surrounding the active site of the enzyme. Mutantsresistant to antiretroviral agents have been detected at low levelsbefore the initiation of therapy. (Mohri H, Singh M K, Ching W T W, etal. (1993) Proc Natl Acad Sci USA 90, 25–29) (Nájera I, Richman D D,Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 1479–1488)(Nájera I, Holguin A, Quiñones-Mateu E, et al. (1995) J Virol 69,23–31). However, these mutant strains represent only a small proportionof the total viral load and may have a replication or competitivedisadvantage compared with wild-type virus. (Coffin J M (1995) Science267, 483–489). The selective pressure of antiretroviral therapy providesthese drug-resistant mutants with a competitive advantage and thus theycome to represent the dominant quasi species (Frost S D W, McLean A R(1994) AIDS 8, 323–332) (Kellam P, Boucher C A B, Tijnagal J M G H(1994) J Gen Virol 75, 341–351) ultimately leading to a rebound in viralload in the patient.

Early development of antiretroviral therapy focused on inhibitors ofreverse transcriptase. Both nucleoside and non-nucleoside inhibitors ofthis enzyme showed significant antiviral activity (DeClerq, E. (1992)AIDS Res. Hum. Retrovir. 8:119–134). However, the clinical benefit ofthese drugs had been limited due to drug resistance, limited potency,and host cellular factors (Richman, D. D. (1993) Ann. Rev. Pharm. Tox.32:149–164). Thus inhibitors targeted against a second essential enzymeof HIV were urgently needed.

In 1988, the protease enzyme of HIV was crystallized and itsthree-dimensional structure was determined, (Navia M A, Fitzgerald P MD, McKeever B M, Leu C T, Heimbach J C, Herber W K, Sigal I S, Darke PL, Springer J P (1989) Nature 337:615–620 and Winters M A, Schapiro J M,Lawrence J, Merigan T C (1997) In Abstracts of the InternationalWorkshop on HIV Drug Resistance, Treatment Strategies and Eradication,St. Petersburg, Fla.) allowing for the rapid development of proteaseinhibitors. Initially, it was hypothesized that HIV protease, unlikereverse transcriptase, would be unable to accommodate mutations leadingto drug resistance. This is not the case, and to date over 20 amino acidsubstitutions in the HIV protease have been observed during treatmentwith the currently available protease inhibitors. The genetic pattern ofmutations conferring resistance to these protease inhibitors is complex,and cross-resistance between structurally different compounds occurs.

Protease Inhibitors

HIV protease was classified as an aspartic proteinase on the basis ofputative active-site homology (Toh H, Ono M, Saigo K, Miyata T (1985)Nature 315:691), its inhibition by peptastin (Richards A D, Roberts R,Dunn B M, Graves M C, Kay J (1989) FEBS Lett 247:113), and its crystalstructure (Navia M A, Fitzgerald P M D, McKeever B M, Lau C T, HeimbachJ C, Herber W K, Sigal I S, Darke P L, Springer J P (1989) Nature337:615–620). The enzyme functions as a homodimer composed of twoidentical 99-amino acid chains (Debouck C, Navia M A, Fitzgerald P M D,McKeever B M, Leu C T, Heimbach J C, Herber W K, Sigal I S, Darke P L,Springer J P (1988) Proc. Natl. Acad. Sci. USA 84:8903–8906), with eachchain containing the characteristic Asp-Thr-Gly active-site sequence atpositions 25 to 27 (Toh H, Ono M, Saigo K, Miyata T (1985) Nature315:691).

HIV protease processes gag (p55) and gag-pol (p160) polyprotein productsinto functional core proteins and viral enzymes (Kohl N E, Diehl R E,Rands E, Davis L J, Hanobik M G, Wolanski B, Dixon R A (1991) J. Virol.65:3007–3014 and Kramer R A, Schaber M D, Skalka A M, Ganguly K,Wong-Staal F, Reddy E P (1986) Science 231:1580–1584). During orimmediately after budding, the polyproteins are cleaved by the enzyme atnine different cleavage sites to yield the structural proteins (p17,p24, p7, and p6) as well as the viral enzymes reverse transcriptase,integrase, and protease (Pettit S C, Michael S F, Swanstrom R (1993)Drug Discov. Des. 1:69–83).

An asparagine replacement for aspartic acid at active-site residue 25results in the production of noninfectious viral particles withimmature, defective cores (Huff J R (1991) AIDS J. Med. Chem.34:2305–2314, Kaplan A H, Zack J A, Knigge M, Paul D A, Kempf D J,Norbeck D W, Swanstrom R (1993) J. Virol. 67:4050–4055, Kohl N E, EminiE A, Schleif W A, Davis L J, Heimbach J C, Dixon R A, Scolnik E M, SigalI S (1988) Proc. Natl. Acad. Sci. USA 85:4686–4690, Peng C, Ho B K,Chang T W, Chang N T (1989) J. Virol. 63:2550–2556). Similarly,wild-type virus particles produced by infected cells treated withprotease inhibitors contain unprocessed precursors and are noninfectious(Crawford S, Goff S P (1985) J. Virol. 53:899–907, Gottlinger H G,Sodroski J G, Haseltine W A (1989) Proc. Natl. Acad. Sci. USA86:5781–5785, Katoh I Y, Yoshinaka Y, Rein A, Shibuya M, Odaka T,Oroszlan S (1985) Virology 145:280–292, Kohl N E, Emini E A, Schleif WA, Davis L J, Heimbach J C, Dixon R A, Scolnik E M, Sigal I S (1988)Proc. Natl. Acad. Sci. USA 85:4686–4690, Peng C, Ho B K, Chang T W,Chang N T (1989) J. Virol. 63:2550–2556, Stewart L, Schatz G, Wogt V M(1990) J. Virol. 64:5076–5092). Unlike reverse transcriptase inhibitors,protease inhibitors block the production of infectious virus fromchronically infected cells (Lambert D M, Petteway, Jr. S R, McDanal C E,Hart T K, Leary J J, Dreyer G B, Meek T D, Bugelski P J, Bolognesi D P,Metcalf B W, Matthews T J (1992) Antibicrob. Agents Chemother.36:982–988). Although the viral protease is a symmetric dimer, it bindsits natural substrates or inhibitors asymmetrically (Dreyer, G B, BoehmJ C, Chenera B, DesJarlais R L, Hassell A M, Meek T D, Tomaszek T A J,Lewis M (1993) Biochemistry 32:937–947, Miller M J, Schneider J,Sathyanarayana B K, Toth M V, Marshall G R, Clawson L, Selk L, Kent S B,Wlodawer A (1989) Science 246:1149–1152). These findings together withthe knowledge that amide bonds of proline residues are not susceptibleto cleavage by mammalian endopeptidases gave rise to the first class ofHIV-1 protease inhibitors based on the transition state mimetic concept,with the phenylalanine-proline cleavage site being the criticalnonscissile bond (Roberts N A, Martin J A, Kinchington D, Broadhurst AV, Craig J C, Duncan I B, Galpin S A, Handa B K, Kay J, Krohn A, LambertR W, Merett J H, Mills J S, Parkes K E B, Redshaw S, Ritchie A J, TaylorD L, Thomas G J, Machin P J (1990) Science 248:358–361).

Amino Acids Implicated in Resistance to Protease Inhibitors.

As new protease inhibitors are developed, the ability of certain aminoacid substitutions to confer resistance to the inhibitor is usuallydetermined by several methods, including selection of resistant strainsin vitro, site-directed mutagenesis, and determination of amino acidchanges that are selected during early phase clinical trials in infectedpatients. While some amino acid substitutions are specificallycorrelated with resistance to certain protease inhibitors (see below),there is considerable overlap between sets of mutations implicated inresistance to all approved protease inhibitors. Many investigators haveattempted to classify these mutations as either being “primary” or“secondary”, with varying definitions. For example, some investigatorsclassify as primary mutations which are predicted, based on X-raycrystallographic data, to be in the enzyme active site with thepotential for direct contact with the inhibitor. (e.g. D3ON, G48V, I50V,V82A/F/S/T, I84V, N88S, L90M). Secondary mutations are usuallyconsidered as being compensatory for defects in enzyme activity imposedby primary mutations, or as having enhancing effects on the magnitude ofresistance imparted by the primary mutations (e.g. L10I/F/R/V,K20I/M/R/T, L24I, V32I, L33F/V, M36I/L/V, M46I/L/V, I47V, I54L/V, L63X,A71T/V, G73A/S/T, V77I, N88D). Lists of mutations and correspondinginhibitors are maintained by several organizations, for example:Schinazi et al., Mutations in retroviral genes associated with drugresistance, Intl. Antiviral News 1999,7:46–69 and Shafer et al., HumanImmunodeficiency Virus Reverse Transcriptase and Protease SequenceDatabase, Nucleic Acids Research 1999, 27(1), 348–352.

Saquinavir

Saquinavir, developed by Hoffmann-La Roche, was the first proteaseinhibitor to undergo clinical evaluation, demonstrating that HIV-1protease was a valid target for the treatment of HIV infection (JacobsenH, Brun-Vezinet F, Duncan I, Hanggi M, Ott M, Vella S, Weber J, Mous J(1994) J. Virol. 68:2016–2020). Saquinavir is a highly activepeptidomimetic protease inhibitor with a 90% inhibitory concentration(IC90) of 6 nM (id). In vitro, saquinavir can select for variants withone or both of two amino acid substitutions in the HIV-1 protease gene,a valine-for-glycine substitution at position 48 (G48V), amethionine-for-leucine substitution at residue 90 (L90M), and the doublesubstitution G48V-L90M (Eberle J, Bechowsky B, Rose D, Hauser U, vonderHelm K, Guertler L, Nitschko H (1995) AIDS Res. Hum. Retroviruses11:671–676, Jacobsen H, Yasargil K, Winslow D L, Craig J C, Kroehn A,Duncan I B, Mous J (1995) Virology 206:527–534, Turriziani O, AntonelliG, Jacobsen H, Mous J, Riva E, Pistello M, Dianzani F (1994) Acta Virol.38:297–298). In most cases, G48V is the first mutation to appear, andcontinued selection results in highly resistant double-mutant variants.A substitution at either residue results in a 3- to 10-fold decreasedsusceptibility to the inhibitor, whereas the simultaneous occurrence ofboth substitutions causes a more severe loss of susceptibilityof >100-fold (id).

In vivo, saquinavir therapy appears to select almost exclusively formutations at codons 90 and 48 (id, Jacobsen H, Hangi M, Ott M, Duncan IB, Owen S, Andreoni M, Vella S, Mous J (1996) J. Infect. Dis.173:1379–1387, Vella S, Galluzzo C, Giannini G, Pirillo M F, Duncan I,Jacobsen H, Andreoni M, Sarmati L, Ercoli L (1996) Antiviral Res.29:91–93). Saquinavir-resistant variants emerge in approximately 45% ofpatients after 1 year of monotherapy with 1,800 mg daily (Craig I C,Duncan I B, Roberts N A, Whittaker L (1993) In Abstracts of the 9thInternational Conference on AIDS, Berlin, Germany, Duncan I B, JacobsenH, Owen S, Roberts N A (1996) In Abstracts of the 3rd Conference ofRetroviruses and Opportunistic Infections, Washington, D. D., id, MousJ, Brun-Vezinet F, Duncan I B, Haenggi M, Jacobsen H, Vella S (1994) InAbstracts of the 10th International Conference on AIDS, Yokohama,Japan). The frequency of resistance is lower (22%) in patients receivingcombination therapy with zidovudine, zalcitabine, and saquinavir(Collier A C, Coombs R, Schoenfeld D A, Bassett R L, Joseph Timpone M S,Baruch A, Jones M, Facey K, Whitacre C, McAuliffe V J, Friedman H M,Merigan T C, Reichmann R C, Hooper C, Corey L (1996) N. Engl. J. Med.334:1011–1017). In contrast to in vitro-selected virus, where the G48Vmutation is the first step to resistance, the L90M exchange is thepredominant mutation selected in vivo while the G48V (2%) or the doublemutant (<2%) is rarely found (id). In another recent study of in vivoresistance during saquinavir monotherapy no patient was found to harbora G48V mutant virus (Ives K J, Jacobsen H, Galpin S A, Garaev M M,Dorrell L, Mous J, Bragman K, Weber J N (1997 J. Antimicrob. Chemother.39:771–779). Interestingly, Winters et al. (id) observed a higherfrequency of the G48V mutation in patients receiving higher saquinavirdoses as monotherapy. All patients (six of six) who initially developedG48V also acquired a V82A mutation either during saquinavir treatment orafter switching to either indinavir or nelfinavir. An identicalmutational pattern was found in another study during saquinavirmonotherapy (Eastman P S, Duncan I B, Gee C, Race E (1997) In Abstractsof the International Workshop on HIV Drug Resistance, TreatmentStrategies and Eradication, St. Petersburg, Fla.). Some residuesrepresent sites of natural polymorphism of the HIV-1 protease (positions10, 36, 63, and 71) and appear to be correlated to the L90M mutation(id). Another substitution, G73S, has been recently identified and mayplay a role in saquinavir resistance in vivo. Isolates from fivepatients with early saquinavir resistance and those from two patientswith induced saquinavir resistance after a switch of therapy toindinavir carried the G73S and the L90M substitutions Dulioust A,Paulous S, Guillemot L, Boue F, Galanaud P, Clavel F (1997) In Abstractsof the International Workshop on HIV Drug Resistance, TreatmentStrategies and Eradication, St. Petersburg, Fla.).

Ritonavir

Ritonavir, developed by Abbott Laboratories, was the second HIV proteaseinhibitor to be licensed in the United States. Ritonavir is a potent andselective inhibitor of HIV protease that is derived from a C2-symmetric,peptidomimetic inhibitor (Ho D D, Toyoshima T, Mo H, Kempf D J, NorbeckD, Chen C M, Wideburg N E, Burt S K, Erickson J W, Singh M K (1994) J.Virol. 68:2016–2020). In vitro activity has been demonstrated against avariety of laboratory strains and clinical isolates of HIV-1 with IC90sof 70 to 200 nM (Kuroda M J, El-Farrash M A, Cloudhury S, Harada S(1995) Virology 210:212–216.

Resistant virus generated by serial, in vitro passages is associatedwith specific mutations at positions 84, 82, 71, 63, and 46 (MarkowitzM, Mo H, Kempf D J, Norbeck D W, Bhat T N, Erickson J W, Ho D D (1995)J. Virol. 69:701–706). The I84V substitution appeared to be the majordeterminant of resistance, resulting in a 10-fold reduction insensitivity to ritonavir. Addition of the V82F mutation confers an evengreater level of resistance, up to 20-fold. The substitutions M46I,L63P, and A71V, when introduced into the protease coding region ofwild-type NL4–3, did not result in significant changes in drugsusceptibility. Based on replication kinetics experiments, these changesare likely to be compensatory for active-site mutations, restoring theimpaired replicative capacity of the combined V82F and I84V mutations.

Indinavir

Indinavir, developed by Merck & Co., is the third HIV protease inhibitorlicensed in the United States. Indinavir is a potent and selectiveinhibitor of HIV-1 and HIV-2 proteases with Ki values of 0.34 and 3.3nM, respectively (Vacca J p, Dorsey B D, Schleif W A, Levin R B,McDaniel S L, Darke P L, Zugay J, Quintero J C, Blahy O M, Roth E,Sardana V V, Schlabach A J, Graham P I, Condra J H, Gotlib L, Holloway MK, Lin J, Chen L-w, Vastag K, Ostobich D, Anderson PS, Emini E A, Huff JR (1994) Proc. Natl. Acad. Sci. USA 91:4096–4100). The drug acts aspeptidomimetic transition state analogue and belongs to the class ofprotease inhibitors known as HAPA (hydroxyaminopentane amide) compounds(ibid). Indinavir provides enhanced aqueous solubility and oralbioavailability and in cell culture exhibits an IC95 of 50 to 100 nM(Emini E A, Schleif W A, Deutsch P, Condra J H (1996) AntiviralChemother. 4:327–331.

Despite early reports of a lack of in vitro resistance by selection withindinavir (id), Tisdale et al. (Tisdale M, Myers R E, Maschera B, ParryN R, Oliver N M, Blair E D (1995) Antibicrob. Agents Chemother.39:1704–1710) were able to obtain resistant variants during selection inMT-4 cells with substitutions at residues 32, 46, 71, and 82. At leastfour mutations were required to produce a significant loss ofsusceptibility (6.1-fold compared with the wild type). The mutation atposition 71, described as compensatory (Markowitz M, Mo H, Kempf D J,Norbeck D W, Bhat T N, Erickson J W, Ho D D (1995) J. Virol. (id),appeared to contribute phenotypic resistance and also to improve virusgrowth. Emini et al. (id) and Condra et al. (Condra J H, Holder D J,Schleif W A, Blahy O M, Danovich R M, Gabryelski L J, Graham D J, LairdD, Quintero J C, Rhodes A, Robbins H L, Roth E, Shivaprakash M, Yang T,Chodakewitz J A, Deutsch P J, Leavitt R Y, Massari F e, Mellors J W,Squires K E, Steigbigel R T, Teppler H, Emini E A (1995) Nature374:569–571) found by constructing mutant HIV-1 clones that at leastthree mutations at residues 46, 63, and 82 were required for thephenotypic manifestation of resistance with a fourfold loss ofsusceptibility.

Nelfinavir

Nelfinavir, developed by Agouron Pharmaceuticals, is a selective,nonpeptidic HIV-1 protease inhibitor that was designed by proteinstructure-based techniques using iterative protein crystallographicanalysis (Appelt K R, Bacquet J, Bartlett C, Booth C L J, Freer S T,Fuhry M M, Gehring M R, Herrmann S M, Howland E F, Janson C A, Jones TR, Kan C C, Kathardekar V, Lewis K K, Marzoni G P, Mathews D A, Mohr C,Moomaw E W, Morse C A, Oatley S J, Ogden R C, Reddy M R, Reich S H,Schoettlin W S, Smith W W, Varney M D, Villafranca J E, Ward R W, WebberS, Webber S E, Welsh K M, White J (1991) J. Med. Chem. 34:1925–1928). Invitro, nelfinavir was found to be a potent inhibitor of HIV-1 proteasewith a Ki of 2.0 nM (Kaldor S W, Kalish V J, Davies J F, Shetty B V,Fritz J E, Appelt K, Burgess J A, Campanale K M, Chirgadze N Y, ClawsonD K, Dressman B A, Hatch S D, Khalil D A, Kosa M B, Lubbehusen P P,Muesing M A, Patrick A K, Reich S H, Su K S, Tatlock J H (1997) J. Med.Chem. 40:3979–3985). The drug demonstrated antiviral activity againstseveral laboratory and clinical HIV-1 and HIV-2 strains with 50%effective concentrations ranging from 9 to 60 nM (Patick A K, Boritzki TJ, Bloom L A (1997) Antimicrob. Agents Chemother. 41:2159–2164).Nelfinavir exhibits additive-to-synergistic effects when combined withother antiretroviral drugs (Partaledis J A, Yamaguchi A K, Tisdale M,Blair E E, Falcione C, Maschera B, Myers R E, Pazhanisamy S, Futer O,Bullinan A B, Stuver C M, Byrn R A, Livingston D J (1995) J. Virol.69:5228–5235). Preclinical data showed high levels of the drug inmesenteric lymph nodes and the spleen and good oral bioavailability(Shetty B V, Kosa M B, Khalil D A, Webber S (1996) Antimicrob. AgentsChemother. 40:110–114).

In vitro, following 22 serial passages of HIV-1_(NL4-3) in the presenceof nelfinavir, a variant (P22) with a sevenfold reduced susceptibilitywas isolated. After an additional six passages a variant (P28) with a30-fold-decreased susceptibility to nelfinavir was identified (Patick AK, Ho H, Markowitz M, Appelt K, Wu B, Musick L, Kaldor S, Reich S, Ho D,Webber S (1996) Antimicrob. Agents Chemother. 40:292–297). Sequenceanalysis of the protease gene from these variants identified indecreasing frequency the substitutions D30N, A71V, and I84V for the P22variant and mutations M46I, I84V/A, L63P, and A71V for the P28 variant.Antiviral susceptibility testing of recombinant mutant HIV-1_(NL4-3)containing various mutations resulted in a fivefold-increased 90%effective concentration for the I84V and D30N single mutants and theM46I/I84V double mutant, whereas no change in susceptibility wasobserved with M46I, L63P, or A71V alone (ibid).

Amprenavir

Amprenavir is a novel protease inhibitor developed by VertexLaboratories and designed from knowledge of the HIV-1 protease crystalstructure (Kim E E, Baker C T, Dyer M D, Murcko M A, Rao B G, Tung R D,Navia M A (1995) J. Am. Chem. Soc. 117:1181–1182). The drug belongs tothe class of sulfonamide protease inhibitors and has been shown to be apotent inhibitor of HIV-1 and HIV-2, with IC50s of 80 and 340 nM,respectively. The mean IC50 for amprenavir against clinical viralisolates was 12 nM (St. Clair M H, Millard J, Rooney J, Tisdale M, ParryN, Sadler B M, Blum M R, Painter G (1996) Antiviral Res. 29:53–56) HIV-1variants 100-fold resistant to amprenavir have been selected by in vitropassage experiments (id). DNA sequence analysis of the protease of thesevariants revealed a sequential accumulation of point mutations resultingin amino acid substitutions L10F, M46I, I47V, and I50V. The keyresistance mutation in the HIV-1 protease substrate binding site isI50V. As a single mutation it confers a two- to threefold decrease insusceptibility (ibid). The other substitutions did not result in reducedsusceptibility when introduced as single mutations into an HIV-1infectious clone (HXB2). However, a triple protease mutant clonecontaining the mutations M46I, I47V, and I50V was 20-fold lesssusceptible to amprenavir than wild-type virus. The I50V mutation hasnot been frequently reported in resistance studies with other HIVprotease inhibitors. Kinetic characterization of these substitutionsdemonstrated an 80-fold reduction in the inhibition constant (K_(i)) forthe I50V single-mutant protease and a 270-fold-reduced K_(i) for thetriple mutant M46I/I47V/I50V, compared to the wild-type enzyme(Pazhanisamy S, St6uvr C M, Cullinan A B, Margolin N, Rao B G (1996) J.Biol. Chem. 271:17979–17985). The single mutants L10F, M46I, and I47Vdid not display reduced affinity for amprenavir. The catalyticefficiency (k_(cat)/K_(m)) of the I50V mutant was decreased up to25-fold, while the triple mutant M46I/I47V/I50V had a 2-fold-higherprocessing efficiency than the I50V single mutant, confirming thecompensatory role of the M46I-and-I47V mutation. The reduced catalyticefficiency (k_(cat)/K_(m)) for these mutants in processing peptidesappeared to be due to both increased K_(m) and decreased k_(cat) values.

Viral Fitness

The relative ability of a given virus or virus mutant to replicate istermed viral fitness. Fitness is dependent on both viral and hostfactors, including the genetic composition of the virus, the host immuneresponse, and selective pressures such as the presence of anti-viralcompounds. Many drug-resistant variants of HIV-1 are less fit than thewild-type, i.e. they grow more slowly in the absence of drug selection.However, since the replication of the wild-type virus is inhibited inthe presence of drug, the resistant mutant can outgrow it. The reductionin fitness may be a result of several factors including: decreasedability of the mutated enzyme (i.e. PR or RT) to recognize its naturalsubstrates, decreased stability of the mutant protein, or decreasedkinetics of enzymatic catalysis. See Back et al., EMBO J. 15: 4040–4049,1996; Goudsmit et al., J. Virol. 70: 5662–5664, 2996; Maschera et al.,J. Biol. Chem. 271: 33231–33235, 1996; Croteau et al., J. Virol. 71:1089–1096, 1997; Zennou et al., J. Virol. 72: 300–3306, 1998; Harriganet al., J. Virol. 72: 3773–3778, 1998; Kosalaraksa et al., J. Virol. 73:5356–5363, 1999; Gerondelis et al., J. Virol. 73: 5803–5813, 1999. Drugresistant viruses that are less fit than wild type may be less virulenti.e. they may cause damage to the host immune system more slowly than awild type virus. Immunological decline may be delayed after theemergence of drug resistant mutants, compared to the rate ofimmunological decline in an untreated patient. The defect causingreductions in fitness may be partially or completely compensated for bythe selection of viruses with additional amino acid substitutions in thesame protein that bears the drug resistance mutations (for example, seeMartinez-Picado et al., J. Virol. 73:3744–3752, 1999), or in otherproteins which interact with the mutated enzyme. Thus, amino acidssurrounding the protease cleavage site in the gag protein may be alteredso that the site is better recognized by a drug-resistant proteaseenzyme (Doyon et al., J. Virol. 70: 3763–3769, 1996; Zhang et al., J.Virol. 71: 6662–6670, 1997; Mammano et al., J, Virol. 72: 7632–7637,1998).

Integrase

Integration of viral DNA into the host chromosome is a necessary processin the HIV replication cycle (Brown, P. O., 1997, in Retroviruses;Coffin, J. M., Hughes, S. H. & Varmus, H. E., eds., Cold Spring HarborLab. Press, Plainview, N.Y., 161–203). The key steps of DNA integrationare carried out by the viral integrase protein, which, along withprotease and reverse transcriptase, is one of three enzymes encoded byHIV. Combination antiviral therapy with protease and reversetranscriptase inhibitors has demonstrated the potential therapeuticefficacy of antiviral therapy for treatment for AIDS (Vandamme, A. M.,Van Vaerenbergh, K. & De Clerq, E., 1998, Antiviral Chem. Chemother. 9,187–203). However, the ability of HIV to rapidly evolve drug resistance,together with toxicity problems, requires the development of additionalclasses of antiviral drugs. Integrase is an attractive target forantivirals because it is essential for HIV replication and, unlikeprotease and reverse transcriptase, there are no known counterparts inthe host cell. Furthermore, integrase uses a single active site toaccommodate two different configurations of DNA substrates, which mayconstrain the ability of HIV to develop drug resistance to integraseinhibitors. However, unlike protease and reverse transcriptase, forwhich several classes of inhibitors have been developed and cocrystalstructures have been determined, progress with the development ofintegrase inhibitors has been slow. A major obstacle has been theabsence of good lead compounds that can serve as the starting point forstructure-based inhibitor development. Although numerous compounds havebeen reported to inhibit integrase activity in vitro, most of thesecompounds exhibit little specificity for integrase and are not useful aslead compounds (Pommier, Y., Pilon, A. A., Bajaj K, K., Mazumder, A. &Neamati, N., 1997, Antiviral Chem. Chemother 8).

HIV-1 integrase is a 32-kDa enzyme that carries out DNA integration in atwo-step reaction (Brown, P. O., ibid.). In the first step, called 3′processing, two nucleotides are removed from each 3′ end of the viralDNA made by reverse transcription. In the next step, called DNA strandtransfer, a pair of transesterification reactions integrates the ends ofthe viral DNA into the host genome. Integrase is comprised of threestructurally and functionally distinct domains, and all three domainsare required for each step of the integration reaction (Engelman, A.Bushman, F. D. & Craigie, R., 1993, EMBO J. 12, 3269–3275). The isolateddomains form homodimers in solution, and the three-dimensionalstructures of all three separate dimers have been determined (Dyda, F.,Hickman, A. B. Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D.R., 1994, Science 226, 1981–1986; Goldgur, Y. Dyda, Hickman, A. B.,Jenkins, T. M., Craigie, R. & Davies, D. R., 1998, Proc. Natl. Acad.Sci., USA 95, 9150–9154; Maignan, S., Guilloteau, J. P., Zhou-Liu, Q.,Clement-Mella, C. & Mikol, V., 1998, J. Mol. Biol. 282, 259–368; Lodi,P. J., Ernst, J. A., Kuszewski, J., Hickman, A. B., Engelman, A.,Craigie, R., Clore, G. M. & Gronenborn, A. M. 1995 Biochemistry 34,9826–9833; Eijkelenboom, A. P., Lutzke, R. A., Boelens, R., Plasterk, R.H., Kaptein, R. & Hard, K. 1995 Nat. Struct. Biol. 2, 807–810; Cai, M.L., Zheng, R., Caffrey, M., Craigie, R., Clore, G. M. & Gronenborn, A.M., 1997 Nat. Struct. Biol. 4, 839–840). Although little is knownconcerning the organization of these domains in the active complex withDNA substrates, integrase is likely to function as at least a tetramer(Dyda, F., Hickman, A. B. Jenkins, T. M., Engelman, A., Craigie, R. &Davies, D. R., 1994, Science 226, 1981–1986). Extensive mutagenesisstudies mapped the catalytic site to the core domain (residues 50–212),which contains the catalytic residues D64, D116, and E152 (Engelman, A.& Craigie R., 1992, J. Virol. 66, 6361–6369; Kulkosky, J., Jones, K. S.,Katz, R. A., Mack, J. P. & Skalka, A. M., 1992, Mol. Cell Biol 12,2331–2338). The structure of this domain of HIV-1 integrase has beendetermined in several crystal forms (Dyda, F., Hickman, A. B. Jenkins,T. M., Engelman, A., Craigie, R. & Davies, D. R., 1994, Science 226,1981–1986; Goldgur, Y. Dyda, Hickman, A. B., Jenkins, T. M., Craigie, R.& Davies, D. R., 1998, Proc. Natl. Acad. Sci., USA 95, 9150–9154;Maignan, S., Guilloteau, J. P., Zhou-Liu, Q., Clement-Mella, C. & Mikol,V., 1998, J. Mol. Biol. 282, 259–368).

Hazuda et al. (Science 287: 646–650, 2000) have described compounds(termed L-731, 988 and L-708,906) which specifically inhibit thestrand-transfer activity of HIV-1 integrase and HIV-1 replication invitro. Viruses grown in the presence of these inhibitors display reducedinhibitor susceptibility and bear mutations in the integrase codingregion at amino acid positions 66 (T66I), 153 (S153Y), and 154 (M154I).Site-directed mutants of a laboratory strain of HIV-1 (HXB2) with theseamino acid changes confirmed their direct role in conferring reducedintegrase inhibitor susceptibility. In addition some of these mutantsdisplayed delayed growth kinetics, suggesting that viral fitness wasimpaired.

It is an object of this invention to provide a drug susceptibility andresistance test capable of showing whether a viral population in apatient is either more or less susceptible to a given prescribed drug.Another object of this invention is to provide a test that will enablethe physician to substitute one or more drugs in a therapeutic regimenfor viruses that show altered susceptibility to a given drug or drugsafter a course of therapy. Yet another object of this invention is toprovide a test that will enable selection of an effective drug regimenfor the treatment of HIV infections and/or AIDS. Yet another object ofthis invention is to provide the means for identifying alterations inthe drug susceptibility profile of a patient's virus, in particularidentifying changes in susceptibility to protease inhibitors. Stillanother object of this invention is to provide a test and methods forevaluating the biological effectiveness of candidate drug compoundswhich act on specific viruses, viral genes and/or viral proteinsparticularly with respect to alterations in viral drug susceptibilityassociated with protease inhibitors. It is also an object of thisinvention to provide the means and compositions for evaluating HIVantiretroviral drug resistance and susceptibility.

It is an object of this invention to provide a method for measuringreplication fitness which can be adapted to viruses, including, but notlimited to human immunodeficiency virus (HIV), hepadnaviruses (humanhepatitis B virus), flaviviruses (human hepatitis C virus) andherpesviruses (human cytomegalovirus). This and other objects of thisinvention will be apparent from the specification as a whole.

SUMMARY OF THE INVENTION

The present invention relates to methods of monitoring, via phenotypicand genotypic methods the clinical progression of human immunodeficiencyvirus infection and its response to antiviral therapy. The invention isalso based, in part, on the discovery that genetic changes in HIVprotease (PR) which confer changes in susceptibility to antiretroviraltherapy may be rapidly determined directly from patient plasma HIV RNAusing phenotypic or genotypic methods. The methods utilize nucleic acidamplification based assays, such as polymerase chain reaction (PCR).Herein-after, such nucleic acid amplification based assays will bereferred to as PCR based assays. This invention is based in part on thediscovery of mutations at codons 10, 20, 36, 46, 63, 77 and 88 of HIVprotease in PRI treated patients in which the presence of certaincombinations of these mutations correlate with changes in certain PRIsusceptibilities. This invention is also based on the discovery thatsusceptibility to HIV protease antivirals may not be altered even ifprimary mutations are present. Additional mutations at secondarypositions in HIV protease are required for a reduction in virussusceptibility. This invention established for the first time that amutation at position 82 of protease (V82A, F, S, or T) in the absence ofanother primary mutation was not correlated with a reduction in drugsusceptibility. Decreased susceptibility to protease inhibitors, such asindinavir and saquinavir, in viruses containing V82A, F, S or T wasobserved in viruses with additional mutations at secondary positions,such as, 24, 71, 54, 46, 10 and/or 63 as described herein. Decreasedsusceptibility to protease inhibitors, such as indinavir and saquinavir,in viruses containing V82A, F, S or T was also observed in viruses withat least 3 or more additional mutations at secondary positions. Thisinventions also established for the first time that a mutation atposition 90 of protease (L90M) in the absence of another primarymutation was not correlated with a reduction in drug susceptibility.Decreased susceptibility to protease inhibitors, such as indinavir andsaquinavir, in viruses containing L90M was observed in viruses withadditional mutations at secondary positions, such as, 73, 71, 77, and/or10 as described herein. Decreased susceptibility to protease inhibitors,such as indinavir and saquinavir, in viruses containing L90M was alsoobserved in viruses with at least 3 or more additional mutations atsecondary positions. The mutations were found in plasma HIV nucleic acidafter a period of time following the initiation of therapy. Thedevelopment of these mutations, or combinations of these mutations, inHIV PR was found to be an indicator of the development of alterations inphenotypic susceptibility/resistance, which can be associated withvirologic failure and subsequent immunological response.

In one embodiment of the invention, a method of assessing theeffectiveness of protease antiretroviral therapy of an HIV-infectedpatient is provided comprising: (a) collecting a plasma sample from theHIV-infected patient; (b) evaluating whether the plasma sample containsnucleic acid encoding HIV protease having a mutation at primary andsecondary positions; and (c) determining changes in susceptibility to aprotease inhibitor.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a substitution atcodon 88 from asparagine to a serine residue either alone or incombination with one or more mutations at other codons selected from thegroup consisting of 10, 20, 36, 46, 63 and/or 77 or a combinationthereof of HIV PR. A mutation at codon 88 from an asparagine residue toa serine residue (N88S) alone correlates with an increase insusceptibility to amprenavir and a mutation at codon 88 from anasparagine residue to a serine residue in combination with mutations atcodons 63 and/or 77 or a combination thereof correlates with an increasein susceptibility to amprenavir and a decrease in nelfinavir andindinavir susceptibility.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect mutations atcodons 10, 20, 36, 46, 63, 77, and 88 of HIV PR which correlate withchanges in susceptibility to antiretroviral therapy and immunologicresponse. Once mutations at these loci have been detected in a patientundergoing PRI antiretroviral therapy, an alteration in the therapeuticregimen should be considered. The timing at which a modification of thetherapeutic regimen should be made, following the assessment ofantiretroviral therapy using PCR based assays, may depend on severalfactors including the patient's viral load, CD4 count, and priortreatment history.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a substitution atcodon 82 from valine to an alanine (V82A), phenylalanine (V82F), serine(V82S), or threonine (V82T) residue either alone or in combination withone or more mutations at other codons, referred to herein as secondarymutations, selected from the group consisting of 20, 24, 36, 71, 54, 46,63 and/or 10 or a combination thereof of HIV PR. A mutation at codon 82from a valine residue to a alanine, phenylalanine, serine or threoninealone correlates with susceptibility to certain protease inhibitorsincluding indinavir and saquinavir. A mutation at codon 82 from a valineresidue to a alanine, phenylalanine, serine or threonine in combinationwith secondary mutations at codons 24 and/or 71 or 20 and/or 36correlates with a reduction in susceptibility to indinavir andsaquinavir, respectively. A mutation at codon 82 from a valine residueto a alanine, phenylalanine, serine or threonine in combination with atleast 3 secondary mutations correlates with a reduction insusceptibility to indinavir and saquinavir.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a substitution atcodon 90 from leucine to a methionine (L90M) residue either alone or incombination with one or more mutations at other codons, referred toherein as secondary mutations, selected from the group consisting of 73,71, 46 and/or 10 or a combination thereof of HIV PR. A mutation at codon90 from a leucine residue to a methionine alone correlates withsusceptibility to certain protease inhibitors including indinavir andsaquinavir. A mutation at codon 90 from a leucine residue to amethionine in combination with secondary mutations at codons 73 and/or71 or 73, 71 and/or 77 correlates with a reduction in susceptibility toindinavir and saquinavir, respectively. A mutation at codon 90 from aleucine residue to a methionine in combination with at least 3 secondarymutations correlates with a reduction in susceptibility to indinavir andsaquinavir.

In another aspect of the invention there is provided a method forassessing the effectiveness of a protease inhibitor antiretroviral drugcomprising: (a) introducing a resistance test vector comprising apatient-derived segment and an indicator gene into a host cell; (b)culturing the host cell from step (a); (c) measuring expression of theindicator gene in a target host cell wherein expression of the indicatorgene is dependent upon the patient derived segment; and (d) comparingthe expression of the indicator gene from step (c) with the expressionof the indicator gene measured when steps (a)–(c) are carried out in theabsence of the PRI anti-HIV drug, wherein a test concentration of thePRI, anti-HIV drug is presented at steps (a)–(c); at steps (b)–(c) or atstep (c).

This invention also provides a method for assessing the effectiveness ofprotease inhibitor antiretroviral therapy in a patient comprising: (a)developing a standard curve of drug susceptibility for an PRI anti-HIVdrug; (b) determining PRI anti-HIV drug susceptibility in the patientusing the susceptibility test described above; and (c) comparing the PRIanti-HIV drug susceptibility in step (b) with the standard curvedetermined in step (a), wherein a decrease in PRI anti-HIVsusceptibility indicates development of anti-HIV drug resistance in thepatient's virus and an increase in PRI anti-HIV susceptibility indicatesdrug hypersensitivity in the patient's virus.

This invention also provides a method for evaluating the biologicaleffectiveness of a candidate PRI HIV antiretroviral drug compoundcomprising: (a) introducing a resistance test vector comprising apatient-derived segment and an indicator gene into a host cell; (b)culturing the host cell from step (a); (c) measuring expression of theindicator gene in a target host cell wherein expression of the indicatorgene is dependent upon the patient derived segment; and (d) comparingthe expression of the indicator gene from step (c) with the expressionof the indicator gene measured when steps (a)–(c) are carried out in theabsence of the candidate PRI anti-viral drug compound, wherein a testconcentration of the candidate PRI anti-viral drug compound is presentat steps (a)–(c); at steps (b)–(c); or at step (c).

The expression of the indicator gene in the resistance test vector inthe target cell is ultimately dependent upon the action of the HIVenzymes (PR and RT) encoded by the patient-derived segment DNAsequences. The indicator gene may be functional or non-functional.

In another aspect this invention is directed to antiretroviral drugsusceptibility and resistance tests for HIV/AIDS. Particular resistancetest vectors of the invention for use in the HIV/AIDS antiretroviraldrug susceptibility and resistance test are identified.

Yet another aspect of this invention provides for the identification andassessment of the biological effectiveness of potential therapeuticantiretroviral compounds for the treatment of HIV and/or AIDS. Inanother aspect, the invention is directed to a novel resistance testvector comprising a patient-derived segment further comprising one ormore mutations on the PR gene and an indicator gene.

Still another aspect of this invention provides for the identificationand assessment of the fitness of a virus infecting a patient. In anotheraspect, the invention is directed to a novel resistance test vectorcomprising a patient-derived segment further comprising one or moremutations on the PR gene and an indicator gene, enabling the measurementof viral fitness.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Resistance Test Vector. A diagrammatic representation of the resistancetest vector comprising a patient derived segment and an indicator gene.

FIG. 2

Two Cell Assay. Schematic Representation of the Assay. A resistance testvector is generated by cloning the patient-derived segment into anindicator gene viral vector. The resistance test vector is thenco-transfected with an expression vector that produces amphotropicmurine leukemia virus (MLV) envelope protein or other viral or cellularproteins which enable infection. Pseudotyped viral particles areproduced containing the protease (PR) and the reverse transcriptase (RT)gene products encoded by the patient-derived DNA sequences. Theparticles are then harvested and used to infect fresh cells. Usingdefective PR and RT sequences it was shown that luciferase activity isdependent on functional PR and RT. PR inhibitors are added to the cellsfollowing transfection and are thus present during particle maturation.RT inhibitors, on the other hand, are added to the cells at the time ofor prior to viral particle infection. The assay is performed in theabsence of drug and in the presence of drug over a wide range ofconcentrations. Luciferase activity is determined and the percentage (%)inhibition is calculated at the different drug concentrations tested.

FIGS. 3 a–3 c

Examples of phenotypic drug susceptibility profiles. Data are analyzedby plotting the percent inhibition of luciferase activity vs. log 10concentration. This plot is used to calculate the drug concentrationthat is required to inhibit virus replication by 50% (IC50) or by 95%(IC95). Shifts in the inhibition curves towards higher drugconcentrations are interpreted as evidence of drug resistance. FIG. 3 ashows the typical curve of drug susceptibility for the nucleosidereverse transcriptase inhibitor AZT. FIG. 3 b shows the typical curve ofdrug susceptibility for the non-nucleoside reverse transcriptaseinhibitor efavirenz. Finally, FIG. 3 c shows the typical curve of drugsusceptibility for the protease inhibitor indinavir. A reduction in drugsusceptibility (resistance) is reflected in a shift in the drugsusceptibility curve toward higher drug concentrations (to the right) ascompared to a baseline (pre-treatment) sample or a drug susceptiblevirus reference control, such as pNL4-3 or HXB-2, when a baseline sampleis not available.

FIGS. 4 a–e

Phenotypic PRI susceptibility profile: patient 0732. A PCR-basedphenotypic susceptibility assay was carried out giving the phenotypicdrug susceptibility profile showing decreased susceptibility tonelfinavir and indinavir, and increased susceptibility amprenavir. FIG.4 a shows a dose response relationship in subjects treated withsaquinavir. FIG. 4 b shows a dose response relationship in subjectstreated with indinavir. FIG. 4 c shows a dose response relationship insubjects treated with ritonavir. FIG. 4 d shows a dose responserelationship in subjects treated with nelfinavir. Finally, FIG. 4 eshows a dose response relationship in subjects treated with amprenavir.

FIGS. 5 a–e

Phenotypic PRI susceptibility profile of a protease mutant generated bysite-specific oligonucleotide-directed mutagenesis. A PCR-basedphenotypic susceptibility assay was carried out giving the phenotypicdrug susceptibility profile of a virus having substitutions at codons63, 77, and 88 (L63P, V77I, and N88s). The profile demonstratedresistance to both nelfinavir and indinavir, and increasedsusceptibility to amprenavir. FIG. 5 a shows a dose responserelationship in subjects treated with saquinavir. FIG. 5 b shows a doseresponse relationship in subjects treated with indinavir. FIG. 5 c showsa dose response relationship in subjects treated with ritonavir. FIG. 5d shows a dose response relationship in subjects treated withnelfinavir. Finally, FIG. 5 e shows a dose response relationship insubjects treated with amprenavir.

FIG. 6.

Two Cell Fitness Assay. Schematic Representation of the Fitness Assay. Afitness test vector is generated by cloning the patient-derived segmentinto an indicator gene viral vector. The fitness test vector is thenco-transfected with an expression vector that produces amphotropicmurine leukemia virus (MLV) envelope protein or other viral or cellularproteins which enable infection. Pseudotyped viral particles areproduced containing the protease (PR) and the reverse transcriptase (RT)gene products encoded by the patient-derived DNA sequences. Theparticles are then harvested and used to infect fresh cells. Usingdefective PR and RT sequences it was shown that luciferase activity isdependent on functional PR and RT. The fitness assay is typicallyperformed in the absence of drug. If desired, the assay can also beperformed at defined drug concentrations. Luciferase activity producedby patient derived viruses is compared to the luciferase activityproduced by well-characterized reference viruses. Replication fitness isexpressed as a percent of the reference.

FIG. 7

Determining the replication fitness of patient viruses. Virus stocksproduced from fitness test vectors derived from patient samples wereused to infect cells. Luciferase activity was measured at various timesafter infection. Patient derived viruses may produce more, approximatelythe same, or less luciferase activity than the reference virus (Ref) andare said to have greater, equivalent, or reduced replication fitness,respectively. The drug susceptibility profiles of three representativepatient derived viruses are shown (P1, P2, P3).

FIG. 8

Identifying alterations in protease or reverse transcriptase functionassociated with differences in replication fitness of patient viruses.Replication fitness is expressed as a percent of the reference virus(top). Fitness measurements are compared to protease processing of thep55 gag polyprotein (middle) and reverse transcriptase activity(bottom). Protease processing is measured by Western blot analysis usingan antibody that reacts with the mature capsid protein (p24). Thedetection of unprocessed p55 or incompletely processed p41 polyproteinsare indicators of reduced cleavage. Reverse transcriptase activity ismeasured using a quantitative RT-PCR assay and is expressed as a percentof the reference virus.

FIG. 9

Correlating reduced replication fitness with reduced reversetranscriptase activity. Viruses containing various amino acidsubstitutions at position 190 (A, S, C, Q, E, T, V) of reversetranscriptase were constructed using site directed mutagenesis. Thereference virus contains G at this position. Replication fitness andreverse transcriptase activities were compared.

FIG. 10

Correlating reduced replication fitness with reduced protease processingof p55 gag. Viruses containing various amino acid substitutions inprotease (D30N, L90M, etc) were constructed using site directedmutagenesis. Replication fitness and p55 gag processing were compared.

FIG. 11

Correlating reduced replication fitness with reduced drugsusceptibility. A large collection (n=134) of patient samples wereevaluated for phenotypic drug susceptibility and replication fitness.Replication fitness and drug susceptibility were compared.

FIG. 12

Relationship between protease inhibitor susceptibility and replicationfitness. Patient samples were sorted based on their replication fitness(<25% of reference, 26–75% of reference, and >75% of reference). Meanvalues for protease inhibitor susceptibility were determined for eachfitness group and plotted for each drug and all drugs combined.

FIG. 13

Relationship between reverse transcriptase inhibitor susceptibility andreplication fitness. Patient samples were sorted based on theirreplication fitness (<25% of reference, 26–75% of reference, and >75% ofreference). Mean values for reverse transcriptase susceptibility weredetermined for each fitness group and plotted for each drug and alldrugs combined.

FIG. 14

Reduced replication fitness is associated with high numbers of proteasemutations, and the L90M mutation. Patient viruses were sorted based onthe number of protease mutations. Viruses with large numbers of proteasemutations or the L90M protease mutation generally exhibit reducedreplication fitness.

FIG. 15

Low replication capacity is associated with specific protease mutations.Patient viruses were sorted based on replication capacity. Specificprotease mutations either alone (D30N) or in combination (L90M plusothers) were observed with high frequency in viruses with reducedreplication fitness.

FIG. 16

Relationship between nelfinavir susceptibility, protease processing andreplication fitness. Patient viruses were sorted based on nelfinavirsusceptibility (<10 or >10 of reference). Protease processing andreplication fitness were plotted for all patient viruses. Viruses withreduced nelfinavir susceptibility generally exhibited reduced proteaseprocessing and reduced replication fitness.

FIG. 17 Protease mutations associated with reduced protease processing.Patient viruses were sorted based on protease processing. Specificprotease mutations were observed at high frequency in viruses withreduced protease processing.

FIG. 18

Representative patient sample exhibiting reversion to drugsusceptibility during a period of drug treatment interruption. Virussamples were collected weekly during a period of treatment interruptionand evaluated for phenotypic drug susceptibility. Values shown representfold change in susceptibility compared to the reference virus.

FIG. 19

Representative patient sample exhibiting increased replication fitnessduring a period of drug treatment interruption. Virus samples werecollected weekly during a period of treatment interruption and evaluatedfor phenotypic drug susceptibility. Fitness values shown representpercent of the reference virus. The increase in fitness between week 9and week 10 corresponds to improved protease processing (bottom) andreversion of the drug resistant phenotype to a drug sensitive phenotype(Figure M).

FIG. 20

Increased replication fitness during treatment interruption. Replicationfitness was measured at the time of treatment interruption and varioustimes during the period of treatment interruption. Generally,replication fitness was significantly higher in samples thatcorresponded to timepoints after the virus had reverted from a drugresistant phenotype to a drug sensitive phenotype.

FIG. 21

FIG. 21 shows a way to measure the replication capacity ofpatient-derived recombinant viruses.

FIG. 22

FIG. 22 shows a way to measure the replication capacity ofpatient-derived recombinant viruses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of monitoring the clinicalprogression of HIV infection in patients receiving antiretroviraltherapy, particularly protease inhibitor antiretroviral therapy.

In one embodiment, the present invention provides for a method ofevaluating the effectiveness of antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV PR having a mutation at one or more positionsin the PR. The mutation(s) correlate positively with alterations inphenotypic susceptibility.

In a specific embodiment, the invention provides for a method ofevaluating the effectiveness of PRI antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV PR having a mutation at codon 88 from anasparagine residue to a serine residue (N88S). This inventionestablished, using a phenotypic susceptibility assay, that a mutation atcodon 88 to a serine residue of HIV protease is correlated with anincrease in amprenavir susceptibility.

In a specific embodiment, the invention provides for a method ofevaluating the effectiveness of PRI antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV PR having a mutation at codon 88 from anasparagine residue to a serine residue (N88S) either alone or incombination with mutations at codons 63 and/or 77 or a combinationthereof. This invention established, using a phenotypic susceptibilityassay, that a mutation at codon 88 to a serine residue of HIV proteaseis correlated with an increase in amprenavir susceptibility and amutation at codon 88 to a serine residue in combination with mutationsat codons 63 and/or 77 or a combination thereof of HIV protease arecorrelated with an increase in amprenavir susceptibility and a decreasein nelfinavir and indinavir susceptibility.

In a specific embodiment, the invention provides for a method ofevaluating the effectiveness of PRI antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV PR having a mutation at codon 88 from anasparagine residue to a serine residue (N88S) either alone or incombination with mutations at codons 46, 63 and/or 77 or a combinationthereof. This invention established, using a phenotypic susceptibilityassay, that a mutation at codon 88 to a serine residue of HIV proteaseis correlated with an increase in amprenavir susceptibility and amutation at codon 88 to a serine residue in combination with mutationsat codons 46, 63 and/or 77 or a combination thereof of HIV protease arecorrelated with an increase in amprenavir susceptibility and a decreasein nelfinavir and indinavir susceptibility.

In a specific embodiment, the invention provides for a method ofevaluating the effectiveness of PRI antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV PR having a mutation at codon 88 from anasparagine residue to a serine residue (N88S) either alone or incombination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or acombination thereof. This invention established, using a phenotypicsusceptibility assay, that a mutation at codon 88 to a serine residue ofHIV protease is correlated with an increase in amprenavir susceptibilityand a mutation at codon 88 to a serine residue in combination withmutations at codons 10, 20, 36, 46, 63 and/or 77 or a combinationthereof of HIV protease are correlated with an increase in amprenavirsusceptibility and a decrease in nelfinavir and indinavirsusceptibility.

Under the foregoing circumstances, the phenotypic susceptibility profileand genotypic profile of the HIV virus infecting the patient has beenaltered reflecting a change in response to the antiretroviral agent. Inthe case of PRI antiretroviral therapy, the HIV virus infecting thepatient may be resistant to one or more PRI s but hypersensitive toanother of the PRI s as described herein. It therefore may be desirableafter detecting the mutation(s), to either increase the dosage of theantiretroviral agent, change to another antiretroviral agent, or add oneor more additional antiretroviral agents to the patient's therapeuticregimen. For example, if the patient was being treated with nelfinavirwhen the N88S mutation arose, the patient's therapeutic regimen maydesirably be altered by either (i) changing to a different PRIantiretroviral agent, such as saquinavir, ritonavir or amprenavir andstopping nelfinavir treatment; or (ii) increasing the dosage ofnelfinavir; or (iii) adding another antiretroviral agent to thepatient's therapeutic regimen. The effectiveness of the modification intherapy may be further evaluated by monitoring viral burden such as byHIV RNA copy number. A decrease in HIV RNA copy number correlatespositively with the effectiveness of a treatment regimen.

The phrase “correlates positively,” as used herein, indicates that aparticular result renders a particular conclusion more likely than otherconclusions.

When reference is made to a particular codon number, it is understoodthat the codon number refers to the position of the amino acid that thecodon codes for. Therefore a codon referencing a particular number isequivalent to a “postion” referencing a particular number, such as forexample, “codon 88” or “position 88”.

Another preferred, non-limiting, specific embodiment of the invention isas follows: A method of evaluating the effectiveness of PRI therapy of apatient comprising (i) collecting a biological sample from anHIV-infected patient; (ii) purifying and converting the viral RNA tocDNA and amplifying HIV sequences using HIV primers that result in a PCRproduct that comprises the PR gene; (iii) performing PCR using primersthat result in PCR products comprising wild type or serine at codon 88;and (iv) determining, via the products of PCR, the presence or absenceof a serine residue at codon 88.

Another preferred, non-limiting, specific embodiment of the invention isas follows: A method of evaluating the effectiveness of PRI therapy of apatient comprising (i) collecting a biological sample from anHIV-infected patient; (ii) purifying and converting the viral RNA tocDNA and amplifying HIV sequences using HIV primers that result in a PCRproduct that comprises the PR gene; (iii) performing PCR using primersthat result in PCR products comprising wild type or serine at codon 88and mutations at codons 63 and/or 77; and (iv) determining, via theproducts of PCR, the presence or absence of a serine residue at codon 88and the presence or absence of mutations at codons 63 and/or 77.

Another preferred, non-limiting, specific embodiment of the invention isas follows: A method of evaluating the effectiveness of PRI therapy of apatient comprising (i) collecting a biological sample from anHIV-infected patient; (ii) purifying and converting the viral RNA tocDNA and amplifying HIV sequences using HIV primers that result in a PCRproduct that comprises the PR gene; (iii) performing PCR using primersthat result in PCR products comprising wild type or serine at codon 88and mutations at codons 63, 77 and/or 46 or a combination thereof; and(iv) determining, via the products of PCR, the presence or absence of aserine residue at codon 88 and the presence or absence of mutations atcodons 63, 77 and/or 46 or a combination thereof.

Another preferred, non-limiting, specific embodiment of the invention isas follows: A method of evaluating the effectiveness of PRI therapy of apatient comprising (i) collecting a biological sample from anHIV-infected patient; (ii) purifying and converting the viral RNA tocDNA and amplifying HIV sequences using HIV primers that result in a PCRproduct that comprises the PR gene; (iii) performing PCR using primersthat result in PCR products comprising wild type or serine at codon 88and mutations at codons 63, 77, 46, 10, 20, and/or 36 or a combinationthereof; and (iv) determining, via the products of PCR, the presence orabsence of a serine residue at codon 88 and the presence or absence ofmutations at codons 63, 77, 46, 10, 20, and/or 36 or a combinationthereof.

The presence of the mutation at codon 88 to a serine of HIV PR indicatesthat the effectiveness of the current or prospective PRI therapy mayrequire alteration, since as shown by this invention mutation at codon88 to a serine residue increases the susceptibility to amprenavir. Usingthe methods of this invention, changes in the PRI therapy would beindicated.

The presence of the mutation at codon 88 to a serine of alone or incombination with mutations at condons 63, 77, 46, 10, 20, and/or 36 or acombination thereof of HIV PR indicates that the effectiveness of thecurrent or prospective PRI therapy may require alteration, since asshown by this invention a mutation at codon 88 to a serine residue aloneincreases the susceptibility to amprenavir and a mutation at codon 88 toa serine residue in combination with mutations at condons 63, 77, 46,10, 20, and/or 36 or a combination increases the susceptibility toamprenavir but also reduces the susceptibility to nelfinavir andindinavir. Using the methods of this invention, changes in the PRItherapy would be indicated.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of evaluating the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVprotease having a mutation at codon 88 to serine. Using the phenotypicsusceptibility assay, it was observed that the presence of the mutationat codon 88 to serine of HIV PR causes a an increase in amprenavirsusceptibility.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of evaluating the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVprotease having a mutation at codon 88 to serine and additionalmutation(s) at codons 63 and/or 77 or a combination thereof. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutation at codon 88 to serine of HIV PR causes an increase inamprenavir susceptibility and the presence of the mutations at codon 88to serine in combination with a mutation at codon(s) 63 and/or 77 or acombination thereof of HIV PR causes a decrease in nelfinavir andindinavir susceptibility while increasing amprenavir susceptibility.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of evaluating the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVprotease having a mutation at codon 88 to serine and additionalmutation(s) at codons 63, 77 and/or 46 or a combination thereof. Usingthe phenotypic susceptibility assay, it was observed that the presenceof the mutation at codon 88 to serine of HIV PR causes an increase inamprenavir susceptibility and the presence of the mutations at codon 88to serine in combination with a mutation at codon(s) 46, 63 and/or 77 ora combination thereof of HIV PR causes a decrease in nelfinavir andindinavir susceptibility while increasing amprenavir susceptibility.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of evaluating the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVprotease having a mutation at codon 88 to serine and additionalmutation(s) at codons 63, 77, 46, 10, 20 and/or 36 or a combinationthereof. Using the phenotypic susceptibility assay, it was observed thatthe presence of the mutation at codon 88 to serine of HIV PR causes anincrease in amprenavir susceptibility and the presence of the mutationsat codon 88 to serine in combination with a mutation at codon(s) 63, 77,46, 10, 20 and/or 36 or a combination thereof of HIV PR causes adecrease in nelfinavir and indinavir susceptibility while increasingamprenavir susceptibility.

This invention also provides the means and methods to use the resistancetest vector comprising an HIV gene and further comprising a PR mutationfor drug screening. More particularly, the invention describes theresistance test vector comprising the HIV protease having a mutation atcodon 88 to a serine alone or in combination with mutations at codons10, 20, 36, 46, 63 and/or 77 or a combination thereof for drugscreening. The invention further relates to novel vectors, host cellsand compositions for isolation and identification of the HIV-1 proteaseinhibitor resistant mutant and using such vectors, host cells andcompositions to carry out anti-viral drug screening. This invention alsorelates to the screening of candidate drugs for their capacity toinhibit said mutant.

This invention provides a method for identifying a compound which iscapable of affecting the function of the protease of HIV-1 comprisingcontacting the compound with the polypeptide(s) comprising all or partof the HIV-1 protease, wherein codon 88 is changed to a serine residue,wherein a positive binding indicates that the compound is capable ofaffecting the function of said protease.

This invention also provides a method for assessing the viral fitness ofpatient's virus comprising: (a) determining the luciferase activity inthe absence of drug for the reference control using the susceptibilitytest described above; (b) determining the luciferase activity in theabsence of drug for the patient virus sample using the susceptibilitytest described above; and (c) comparing the luciferase activitydetermined in step (b) with the luciferase activity determined in step(a), wherein a decrease in luciferase activity indicates a reduction inviral fitness of the patient's virus.

If a resistance test vector is constructed using a patient derivedsegment from a patient virus which is unfit, and the fitness defect isdue to genetic alterations in the patient derived segment, then thevirus produced from cells transfected with the resistance test vectorwill produce luciferase more slowly. This defect will be manifested asreduced luciferase activity (in the absence of drug) compared to thedrug sensitive reference control, and may be expressed as a percentageof the control.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect mutations atpositions 20 and 88 of HIV PR, which correlate with a reduction in viralfitness and immunological response.

It is a further embodiment of this invention to provide a means andmethod for measuring replication fitness for viruses, including, but notlimited to human immunodeficiency virus (HIV), hepadnaviruses (humanhepatitis B virus), flaviviruses (human hepatitis C virus) andherpesviruses (human cytomegalovirus).

This invention further relates to a means and method for measuring thereplication fitness of HIV-1 that exhibits reduced drug susceptibilityto reverse transcriptase inhibitors and protease inhibitors.

In a further embodiment of the invention, a means and methods areprovided for measuring replication fitness for other classes ofinhibitors of HIV-1 replication, including, but not limited tointegration, virus assembly, and virus attachment and entry.

This invention relates to a means and method for identifying mutationsin protease or reverse transcriptase that alter replication fitness.

In a further embodiment of the invention, a means and methods areprovided for identifying mutations that alter replication fitness forother components of HIV-1 replication, including, but not limited tointegration, virus assembly, and virus attachment and entry.

This invention also relates to a means and method for quantifying theaffect that specific mutations in protease or reverse transcriptase haveon replication fitness.

In a further embodiment of the invention, a means and method areprovided for quantifying the affect that specific protease and reversetranscriptase mutations have on replication fitness in other viral genesinvolved in HIV-1 replication, including, but not limited to the gag,pol, and envelope genes.

This invention also relates to the high incidence of patient sampleswith reduced replication fitness.

This invention relates to the correlation between reduced drugsusceptibility and reduced replication fitness.

This invention further relates to the occurrence of viruses with reducedfitness in patients receiving protease inhibitor and/or reversetranscriptase inhibitor treatment.

This invention further relates to the incidence of patient samples withreduced replication fitness in which the reduction in fitness is due toaltered protease processing of the gag polyprotein (p55).

This invention further relates to the incidence of protease mutations inpatient samples that exhibit low, moderate or normal (wildtype)replication fitness.

This invention further relates to protease mutations that are frequentlyobserved, either alone or in combination, in viruses that exhibitreduced replication capacity.

This invention also relates to the incidence of patient samples withreduced replication fitness in which the reduction in fitness is due toaltered reverse transcriptase activity. This invention relates to theoccurrence of viruses with reduced replication fitness in patientsfailing antiretroviral drug treatment. This invention further relates toa means and method for using replication fitness measurements to guidethe treatment of HIV-1. This invention further relates to a means andmethod for using replication fitness measurements to guide the treatmentof patients failing antiretroviral drug treatment. This inventionfurther relates to the means and methods for using replication fitnessmeasurements to guide the treatment of patients newly infected withHIV-1.

This invention, provides the means and methods for using replicationfitness measurements to guide the treatment of viral diseases,including, but not limited to HIV-1, hepadnaviruses (human hepatitis Bvirus), flaviviruses (human hepatitis C virus) and herpesviruses (humancytomegalovirus).

In a further embodiment, the invention provides a method for determiningreplication capacity for a patient's virus comprising:

-   -   (a) introducing a resistance test vector comprising a patient        derived segment and an indicator gene into a host cell;    -   (b) culturing the host cell from (a);    -   (c) harvesting viral particles from step (b) and infecting        target host cells;    -   (d) measuring expression of the indicator gene in the target        host cell, wherein the expression of the indicator gene is        dependent upon the patient-derived segment;    -   (e) comparing the expression of the indicator gene from (d) with        the expression of the indicator gene measured when steps (a)        through (d) are carried out in a control resistance test vector;        and    -   (f) normalizing the expression of the indicator gene by        measuring an amount of virus in step (c).

As used herein, “patient-derived segment” encompasses segments derivedfrom human and various animal species. Such species include, but are notlimited to chimpanzees, horses, cattles, cats and dogs.

Patient-derived segments can also be incorporated into resistance testvectors using any of several alternative cloning techniques as set forthin detail in U.S. Pat. No. 5,837,464 (International Publication NumberWO 97/27319) which is hereby incorporated by reference. For example,cloning via the introduction of class II restriction sites into both theplasmid backbone and the patient-derived segments or by uracil DNAglycosylase primer cloning.

The patient-derived segment may be obtained by any method of molecularcloning or gene amplification, or modifications thereof, by introducingpatient sequence acceptor sites, as described below, at the ends of thepatient-derived segment to be introduced into the resistance testvector. For example, in a gene amplification method such as PCR,restriction sites corresponding to the patient-sequence acceptor sitescan be incorporated at the ends of the primers used in the PCR reaction.Similarly, in a molecular cloning method such as cDNA cloning, saidrestriction sites can be incorporated at the ends of the primers usedfor first or second strand cDNA synthesis, or in a method such asprimer-repair of DNA, whether cloned or uncloned DNA, said restrictionsites can be incorporated into the primers used for the repair reaction.The patient sequence acceptor sites and primers are designed to improvethe representation of patient-derived segments. Sets of resistance testvectors having designed patient sequence acceptor sites providerepresentation of patient-derived segments that may be underrepresentedin one resistance test vector alone.

“Resistance test vector” means one or more vectors which taken togethercontain DNA comprising a patient-derived segment and an indicator gene.Resistance test vectors are prepared as described in U.S. Pat. No.5,837,464 (International Publication Number WO 97/27319), which ishereby incorporated by reference, by introducing patient sequenceacceptor sites, amplifying or cloning patient-derived segments andinserting the amplified or cloned sequences precisely into indicatorgene viral vectors at the patient sequence acceptor sites.Alternatively, a resistance test vector (also referred to as aresistance test vector system) is prepared by introducing patientsequence acceptor sites into a packaging vector, amplifying or cloningpatient-derived segments and inserting the amplified or cloned sequencesprecisely into the packaging vector at the patient sequence acceptorsites and co-transfecting this packaging vector with an indicator geneviral vector.

“Indicator or indicator gene,” as described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319) refers to a nucleic acidencoding a protein, DNA or RNA structure that either directly or througha reaction gives rise to a measurable or noticeable aspect, e.g. a coloror light of a measurable wavelength or in the case of DNA or RNA used asan indicator a change or generation of a specific DNA or RNA structure.Preferred examples of an indicator gene is the E. coli lacZ gene whichencodes beta-galactosidase, the luc gene which encodes luciferase eitherfrom, for example, Photonis pyralis (the firefly) or Renilla reniformis(the sea pansy), the E. coli phoA gene which encodes alkalinephosphatase, green fluorescent protein and the bacterial CAT gene whichencodes chloramphenicol acetyltransferase. The indicator or indicatorgene may be functional or non-functional as described in U.S. Pat. No.5,837,464 (International Publication Number WO 97/27319).

The phenotypic drug susceptibility and resistance tests of thisinvention may be carried out in one or more host cells as described inU.S. Pat. No. 5,837,464 (International Publication Number WO 97/27319)which is incorporated herein by reference. Viral drug susceptibility isdetermined as the concentration of the anti-viral agent at which a givenpercentage of indicator gene expression is inhibited (e.g. the IC50 foran anti-viral agent is the concentration at which 50% of indicator geneexpression is inhibited). A standard curve for drug susceptibility of agiven anti-viral drug can be developed for a viral segment that iseither a standard laboratory viral segment or from a drug-naive patient(i.e. a patient who has not received any anti-viral drug) using themethod described in the aforementioned patent. Correspondingly, viraldrug resistance is a decrease in viral drug susceptibility for a givenpatient compared to such a given standard or when making one or moresequential measurements in the same patient over time, as determined bydecreased susceptibility in virus from later time points compared tothat from earlier time points.

The antiviral drugs being added to the test system are added at selectedtimes depending upon the target of the antiviral drug. For example, inthe case of HIV protease inhibitors, including saquinavir, ritonavir,indinavir, nelfinavir and amprenavir, they are added to packaging hostcells at the time of or shortly after their transfection with aresistance test vector, at an appropriate range of concentrations. HIVreverse transcriptase inhibitors, including AZT, ddI, ddC, d4T, 3TC,abacavir, nevirapine, delavirdine and efavirenz are added to target hostcells at the time of or prior to infection by the resistance test vectorviral particles, at an appropriate range of concentration.Alternatively, the antiviral drugs may be present throughout the assay.The test concentration is selected from a range of concentrations whichis typically between about 8×10⁻⁶ μM and about 2 mM and morespecifically for each of the following drugs: saquinavir, indinavir,nelfinavir and amprenavir, from about 2.3×10⁻⁵ μM to about 1.5 μM andritonavir, from about 4.5×10⁻⁵ μM to about 3 μM.

In another embodiment of this invention, a candidate PRI antiretroviralcompound is tested in the phenotypic drug susceptibility and resistancetest using the resistance test vector comprising PR having a mutation atcodon 88 to a serine. The candidate antiviral compound is added to thetest system at an appropriate range of concentrations and at thetransfection step. Alternatively, more than one candidate antiviralcompound may be tested or a candidate antiviral compound may be testedin combination with an approved antiviral drug such as AZT, ddI, ddC,d4T, 3TC, abacavir, delavirdine, nevirapine, efavirenz, saquinavir,ritonavir, indinavir, nelfinavir, amprenavir, or a compound which isundergoing clinical trials such as adefovir and ABT-378. Theeffectiveness of the candidate antiviral will be evaluated by measuringthe expression or inhibition of the indicator gene. In another aspect ofthis embodiment, the drug susceptibility and resistance test may be usedto screen for viral mutants. Following the identification of mutantsresistant to either known antiretrovirals or candidate antiretroviralsthe resistant mutants are isolated and the DNA is analyzed. A library ofviral resistant mutants can thus be assembled enabling the screening ofcandidate PRI antiretrovirals, alone or in combination. This will enableone of ordinary skill to identify effective PRI antiretrovirals anddesign effective therapeutic regimens.

In another embodiment of this invention, a method of assessing theeffectiveness of protease antiretroviral therapy of an HIV-infectedpatient is provided comprising:

(a) collecting a biological sample from the HIV-infected patient;

(b) evaluating whether the biological sample contains nucleic acidencoding HIV protease having a mutation at codon 82 or codon 90; and

(c) determining changes in susceptibility to protease inhibitors.

In another embodiment of this invention, the method is provided, whereinstep (c) determines changes in susceptibility to saquinavir.

In another embodiment of this invention, the method is provided, whereinthe mutation at codon 82 codes for alanine (A), phenylalanine (F),serine (S), or threonine (T).

In another embodiment of this invention, the method is provided, whereinthe mutation at codon 82 is a substitution of alanine (A), phenylalanine(F), serine (S), or threonine (T) for valine (V).

In another embodiment of this invention, the method is provided, whereinthe mutation at codon 90 codes for methionine (M).

In another embodiment of this invention, the method is provided, whereinthe mutation at codon 90 is a substitution of methionine (M) for leucine(L).

In another embodiment of this invention, a method for evaluating thebiological effectiveness of a candidate HIV protease antiretroviral drugcompound is provided comprising:

-   -   (a) introducing a resistance test vector comprising a        patient-derived segment having nucleic acid encoding HIV        protease with a mutation at codon 82 or codon 90 and an        indicator gene into a host cell;    -   (b) culturing the host cell from step (a);    -   (c) measuring the indicator gene in a target host cell; and    -   (d) comparing the measurement of the indicator gene from        step (c) with the measurement of the indicator gene measured        when steps (a)–(c) are carried out in the absence of the        candidate antiretroviral drug compound;        wherein a test concentration of the candidate antiretroviral        drug compound is present at steps (a)–(c); at steps (b)–(c); or        at step (c).

In another embodiment of this invention, a resistance test vectorcomprising an HIV patient-derived segment further comprising proteasehaving a mutation at codon 82 or codon 90 and an indicator gene, whereinthe expression of the indicator gene is dependent upon thepatient-derived segment.

In another embodiment of this invention, the resistance test vector isprovided, wherein the patient-derived segment having a mutation at codon82 codes for alanine (A), phenylalanine (F), serine (S), or threonine(T).

In another embodiment of this invention, the resistance test vector ofis provided, wherein the patient-derived segment having a mutation atcodon 82 is a substitution of alanine (A), phenylalanine (F), serine(S), or threonine (T) for valine (V).

In another embodiment of this invention, the resistance test vector isprovided, wherein the patient-derived segment having a mutation at codon90 codes for methionine (M).

In yet another embodiment of this invention, the resistance test vectoris provided, wherein the patient-derived segment having a mutation atcodon 90 is a substitution of methionine (M) for leucine (L).

In another embodiment of this invention, a method for determiningreplication capacity for a patient's virus is provided comprising:

(a) introducing a resistance test vector comprising a patient-derivedsegment and an indicator gene into a host cell;

(b) culturing the host cell from (a);

(c) harvesting viral particles from step (b) and infecting target hostcells;

(d) measuring expression of the indicator gene in the target host cell,wherein the expression of the indicator gene is dependent upon thepatient-derived segment; and

(e) comparing the expression of the indicator gene from (d) with theexpression of the indicator gene measured when steps (a) through (d) arecarried out in a control resistance test vector.

In another embodiment of this invention, the method further comprisesthe step of:

(f) normalizing the expression of the indicator gene by measuring anamount of virus in step (c).

In another embodiment of this invention, the method is provided whereinthe patient-derived segment comprises nucleic acid encoding HIVintegrase having a mutation at codon 66.

In another embodiment of this invention, the method is provided whereinthe patient-derived segment comprises nucleic acid encoding HIVintegrase having a mutation at codon 154.

In another embodiment of this invention, the method is provided whereinthe patient-derived segment comprises nucleic acid encoding HIVintegrase having mutations at codon 66 and codon 153.

In another embodiment of this invention, the method is provided whereinthe patient-derived segment comprises nucleic acid encoding HIVintegrase having mutations at codon 66 and codon 154.

The structure, life cycle and genetic elements of the viruses whichcould be tested in the drug susceptibility and resistance test of thisinvention would be known to one of ordinary skill in the art. It isuseful to the practice of this invention, for example, to understand thelife cycle of a retrovirus, as well as the viral genes required forretrovirus rescue and infectivity. Retrovirally infected cells shed amembrane virus containing a diploid RNA genome. The virus, studded withan envelope glycoprotein (which serves to determine the host range ofinfectivity), attaches to a cellular receptor in the plasma membrane ofthe cell to be infected. After receptor binding, the virus isinternalized and uncoated as it passes through the cytoplasm of the hostcell. Either on its way to the nucleus or in the nucleus, the reversetranscriptase molecules resident in the viral core drive the synthesisof the double-stranded DNA provirus, a synthesis that is primed by thebinding of a tRNA molecule to the genomic viral RNA. The double-strandedDNA provirus is subsequently integrated in the genome of the host cell,where it can serve as a transcriptional template for both mRNAs encodingviral proteins and virion genomic RNA, which will be packaged into viralcore particles. On their way out of the infected cell, core particlesmove through the cytoplasm, attach to the inside of the plasma membraneof the newly infected cell, and bud, taking with them tracts of membranecontaining the virally encoded envelope glycoprotein gene product. Thiscycle of infection—reverse transcription, transcription, translation,virion assembly, and budding—repeats itself over and over again asinfection spreads.

The viral RNA and, as a result, the proviral DNA encode severalcis-acting elements that are vital to the successful completion of theviral lifecycle. The virion RNA carries the viral promoter at its 3′end. Replicative acrobatics place the viral promoter at the 5′ end ofthe proviral genome as the genome is reverse transcribed. Just 3′ to the5′ retroviral LTR lies the viral packaging site. The retrovirallifecycle requires the presence of virally encoded transacting factors.The viral-RNA-dependent DNA polymerase (pol)-reverse transcriptase isalso contained within the viral core and is vital to the viral lifecycle in that it is responsible for the conversion of the genomic RNA tothe integrative intermediate proviral DNA. The viral envelopeglycoprotein, env, is required for viral attachment to the uninfectedcell and for viral spread. There are also transcriptionaltrans-activating factors, so called transactivators, that can serve tomodulate the level of transcription of the integrated parental provirus.Typically, replication-competent (non-defective) viruses areself-contained in that they encode all of these trans-acting factors.Their defective counterparts are not self-contained.

In the case of a DNA virus, such as a hepadnavirus, understanding thelife cycle and viral genes required for infection is useful to thepractice of this invention. The process of HBV entry has not been welldefined. Replication of HBV uses an RNA intermediate template. In theinfected cell the first step in replication is the conversion of theasymmetric relaxed circle DNA (rc-DNA) to covalently closed circle DNA(cccDNA). This process, which occurs within the nucleus of infectedliver cells, involves completion of the DNA positive-strand synthesisand ligation of the DNA ends. In the second step, the cccDNA istranscribed by the host RNA polymerase to generate a 3.5 kB RNA template(the pregenome). This pregenome is complexed with protein in the viralcore. The third step involves the synthesis of the first negative-senseDNA strand by copying the pregenomic RNA using the virally encoded Pprotein reverse transcriptase. The P protein also serves as the minusstrand DNA primer. Finally, the synthesis of the second positive-senseDNA strand occurs by copying the first DNA strand, using the P proteinDNA polymerase activity and an oligomer of viral RNA as primer. Thepregenome also transcribes mRNA for the major structural core proteins.

The following flow chart illustrates certain of the various vectors andhost cells which may be used in this invention. It is not intended to beall inclusive.

Host Cells

Packaging Host Cell—transfected with packaging expression vectors

Resistance Test Vector Host Cell—a packaging host cell transfected witha resistance test vector

Target Host Cell—a host cell to be infected by a resistance test vectorviral particle produced by the resistance test vector host cell

Resistance Test Vector

“Resistance test vector” means one or more vectors which taken togethercontain DNA or RNA comprising a patient-derived segment and an indicatorgene. In the case where the resistance test vector comprises more thanone vector the patient-derived segment may be contained in one vectorand the indicator gene in a different vector. Such a resistance testvector comprising more than one vector is referred to herein as aresistance test vector system for purposes of clarity but isnevertheless understood to be a resistance test vector. The DNA or RNAof a resistance test vector may thus be contained in one or more DNA orRNA molecules. In one embodiment, the resistance test vector is made byinsertion of a patient-derived segment into an indicator gene viralvector. In another embodiment, the resistance test vector is made byinsertion of a patient-derived segment into a packaging vector while theindicator gene is contained in a second vector, for example an indicatorgene viral vector. As used herein, “patient-derived segment” refers toone or more viral segments obtained directly from a patient usingvarious means, for example, molecular cloning or polymerase chainreaction (PCR) amplification of a population of patient-derived segmentsusing viral DNA or complementary DNA (cDNA) prepared from viral RNA,present in the cells (e.g. peripheral blood mononuclear cells, PBMC),serum or other bodily fluids of infected patients. When a viral segmentis “obtained directly” from a patient it is obtained without passage ofthe virus through culture, or if the virus is cultured, then by aminimum number of passages to essentially eliminate the selection ofmutations in culture. The term “viral segment” refers to any functionalviral sequence or viral gene encoding a gene product (e.g., a protein)that is the target of an anti-viral drug. The term “functional viralsequence” as used herein refers to any nucleic acid sequence (DNA orRNA) with functional activity such as enhancers, promoters,polyadenylation sites, sites of action of trans-acting factors, such astar and RRE, packaging sequences, integration sequences, or splicingsequences. If a drug were to target more than one functional viralsequence or viral gene product then patient-derived segmentscorresponding to each said viral gene would be inserted in theresistance test vector. In the case of combination therapy where two ormore anti-virals targeting two different functional viral sequences orviral gene products are being evaluated, patient-derived segmentscorresponding to each functional viral sequence or viral gene productwould be inserted in the resistance test vector. The patient-derivedsegments are inserted into unique restriction sites or specifiedlocations, called patient sequence acceptor sites, in the indicator geneviral vector or for example, a packaging vector depending on theparticular construction being used as described herein.

As used herein, “patient-derived segment” encompasses segments derivedfrom human and various animal species. Such species include, but are notlimited to chimpanzees, horses, cattles, cats and dogs.

Patient-derived segments can also be incorporated into resistance testvectors using any of several alternative cloning techniques. Forexample, cloning via the introduction of class II restriction sites intoboth the plasmid backbone and the patient-derived segments or by uracilDNA glycosylase primer cloning (refs).

The patient-derived segment may be obtained by any method of molecularcloning or gene amplification, or modifications thereof, by introducingpatient sequence acceptor sites, as described below, at the ends of thepatient-derived segment to be introduced into the resistance testvector. For example, in a gene amplification method such as PCR,restriction sites corresponding to the patient-sequence acceptor sitescan be incorporated at the ends of the primers used in the PCR reaction.Similarly, in a molecular cloning method such as cDNA cloning, saidrestriction sites can be incorporated at the ends of the primers usedfor first or second strand cDNA synthesis, or in a method such asprimer-repair of DNA, whether cloned or uncloned DNA, said restrictionsites can be incorporated into the primers used for the repair reaction.The patient sequence acceptor sites and primers are designed to improvethe representation of patient-derived segments. Sets of resistance testvectors having designed patient sequence acceptor sites providerepresentation of patient-derived segments that would beunderrepresented in one resistance test vector alone.

Resistance test vectors are prepared by modifying an indicator geneviral vector (described below) by introducing patient sequence acceptorsites, amplifying or cloning patient-derived segments and inserting theamplified or cloned sequences precisely into indicator gene viralvectors at the patient sequence acceptor sites.

The resistance test vectors are constructed from indicator gene viralvectors which are in turn derived from genomic viral vectors orsubgenomic viral vectors and an indicator gene cassette, each of whichis described below. Resistance test vectors are then introduced into ahost cell. Alternatively, a resistance test vector (also referred to asa resistance test vector system) is prepared by introducing patientsequence acceptor sites into a packaging vector, amplifying or cloningpatient-derived segments and inserting the amplified or cloned sequencesprecisely into the packaging vector at the patient sequence acceptorsites and co-transfecting this packaging vector with an indicator geneviral vector.

In one preferred embodiment, the resistance test vector may beintroduced into packaging host cells together with packaging expressionvectors, as defined below, to produce resistance test vector viralparticles that are used in drug resistance and susceptibility tests thatare referred to herein as a “particle-based test.” In an alternativepreferred embodiment, the resistance test vector may be introduced intoa host cell in the absence of packaging expression vectors to carry outa drug resistance and susceptibility test that is referred to herein asa “non-particle-based test.” As used herein a “packaging expressionvector” provides the factors, such as packaging proteins (e.g.structural proteins such as core and envelope polypeptides), transactingfactors, or genes required by replication-defective retrovirus orhepadnavirus. In such a situation, a replication-competent viral genomeis enfeebled in a manner such that it cannot replicate on its own. Thismeans that, although the packaging expression vector can produce thetrans-acting or missing genes required to rescue a defective viralgenome present in a cell containing the enfeebled genome, the enfeebledgenome cannot rescue itself.

Indicator or Indicator Gene

“Indicator or indicator gene” refers to a nucleic acid encoding aprotein, DNA or RNA structure that either directly or through a reactiongives rise to a measurable or noticeable aspect, e.g. a color or lightof a measurable wavelength or in the case of DNA or RNA used as anindicator a change or generation of a specific DNA or RNA structure.Preferred examples of an indicator gene is the E. coli lacZ gene whichencodes beta-galactosidase, the luc gene which encodes luciferase eitherfrom, for example, Photonis pyralis (the firefly) or Renilla reniformis(the sea pansy), the E. coli phoA gene which encodes alkalinephosphatase, green fluorescent protein and the bacterial CAT gene whichencodes chloramphenicol acetyltransferase. Additional preferred examplesof an indicator gene are secreted proteins or cell surface proteins thatare readily measured by assay, such as radioimmunoassay (RIA), orfluorescent activated cell sorting (FACS), including, for example,growth factors, cytokines and cell surface antigens (e.g. growthhormone, Il-2 or CD4, respectively). “Indicator gene” is understood toalso include a selection gene, also referred to as a selectable marker.Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR), thymidine kinase, hygromycin, neomycin,zeocin or E. coli gpt. In the case of the foregoing examples ofindicator genes, the indicator gene and the patient-derived segment arediscrete, i.e. distinct and separate genes. In some cases apatient-derived segment may also be used as an indicator gene. In onesuch embodiment in which the patient-derived segment corresponds to morethan one viral gene which is the target of an anti-viral, one of saidviral genes may also serve as the indicator gene. For example, a viralprotease gene may serve as an indicator gene by virtue of its ability tocleave a chromogenic substrate or its ability to activate an inactivezymogen which in turn cleaves a chromogenic substrate, giving rise ineach case to a color reaction. In all of the above examples of indicatorgenes, the indicator gene may be either “functional” or “non-functional”but in each case the expression of the indicator gene in the target cellis ultimately dependent upon the action of the patient-derived segment.

Functional Indicator Gene

In the case of a “functional indicator gene” the indicator gene may becapable of being expressed in a “packaging host cell/resistance testvector host cell” as defined below, independent of the patient-derivedsegment, however the functional indicator gene could not be expressed inthe target host cell, as defined below, without the production offunctional resistance test vector particles and their effectiveinfection of the target host cell. In one embodiment of a functionalindicator gene, the indicator gene cassette, comprising control elementsand a gene encoding an indicator protein, is inserted into the indicatorgene viral vector with the same or opposite transcriptional orientationas the native or foreign enhancer/promoter of the viral vector. Oneexample of a functional indicator gene in the case of HIV or HBV, placesthe indicator gene and its promoter (a CMV IE enhancer/promoter) in thesame or opposite transcriptional orientation as the HIV-LTR or HBVenhancer-promoter, respectively, or the CMV IE enhancer/promoterassociated with the viral vector.

Non-Functional Indicator Gene

Alternatively the indicator gene, may be “non-functional” in that theindicator gene is not efficiently expressed in a packaging host celltransfected with the resistance test vector, which is then referred to aresistance test vector host cell, until it is converted into afunctional indicator gene through the action of one or more of thepatient-derived segment products. An indicator gene is renderednon-functional through genetic manipulation according to this invention.

1. Permuted Promoter In one embodiment an indicator gene is renderednon-functional due to the location of the promoter, in that, althoughthe promoter is in the same transcriptional orientation as the indicatorgene, it follows rather than precedes the indicator gene codingsequence. This misplaced promoter is referred to as a “permutedpromoter.” In addition to the permuted promoter the orientation of thenon-functional indicator gene is opposite to that of the native orforeign promoter/enhancer of the viral vector. Thus the coding sequenceof the non-functional indicator gene can neither be transcribed by thepermuted promoter nor by the viral promoters. The non-functionalindicator gene and its permuted promoter is rendered functional by theaction of one or more of the viral proteins. One example of anon-functional indicator gene with a permuted promoter in the case ofHIV, places a T7 phage RNA polymerase promoter (herein referred to as T7promoter) promoter in the 5′ LTR in the same transcriptional orientationas the indicator gene. The indicator gene cannot be transcribed by theT7 promoter as the indicator gene cassette is positioned upstream of theT7 promoter. The non-functional indicator gene in the resistance testvector is converted into a functional indicator gene by reversetranscriptase upon infection of the target cells, resulting from therepositioning of the T7 promoter, by copying from the 5′ LTR to the 3′LTR, relative to the indicator gene coding region. Following theintegration of the repaired indicator gene into the target cellchromosome by HIV integrase, a nuclear T7 RNA polymerase expressed bythe target cell transcribes the indicator gene. One example of anon-functional indicator gene with a permuted promoter in the case ofHBV, places an enhancer-promoter region downstream or 3′ of theindicator gene both having the same transcriptional orientation. Theindicator gene cannot be transcribed by the enhancer-promoter as theindicator gene cassette is positioned upstream. The non-functionalindicator gene in the resistance test vector is converted into afunctional indicator gene by reverse transcription and circularizationof the HBV indicator gene viral vector by the repositioning of theenhancer-promoter upstream relative to the indicator gene coding region.

A permuted promoter may be any eukaryotic or prokaryotic promoter whichcan be transcribed in the target host cell. Preferably the promoter willbe small in size to enable insertion in the viral genome withoutdisturbing viral replication. More preferably, a promoter that is smallin size and is capable of transcription by a single subunit RNApolymerase introduced into the target host cell, such as a bacteriophagepromoter, will be used. Examples of such bacteriophage promoters andtheir cognate RNA polymerases include those of phages T7, T3 and Sp6. Anuclear localization sequence (NLS) may be attached to the RNApolymerase to localize expression of the RNA polymerase to the nucleuswhere they may be needed to transcribed the repaired indicator gene.Such an NLS may be obtained from any nuclear-transported protein such asthe SV40 T antigen. If a phage RNA polymerase is employed, an internalribosome entry site (IRES) such as the EMC virus 5′ untranslated region(UTR) may be added in front of the indicator gene, for translation ofthe transcripts which are generally uncapped. In the case of HIV, thepermuted promoter itself can be introduced at any position within the 5′LTR that is copied to the 3′ LTR during reverse transcription so long asLTR function is not disrupted, preferably within the U5 and R portionsof the LTR, and most preferably outside of functionally important andhighly conserved regions of U5 and R. In the case of HBV, the permutedpromoter can be placed at any position that does not disrupt the cisacting elements that are necessary for HBV DNA replication. Blockingsequences may be added at the ends of the resistance test vector shouldthere be inappropriate expression of the non-functional indicator genedue to transfection artifacts (DNA concatenation). In the HIV example ofthe permuted T7 promoter given above, such a blocking sequence mayconsist of a T7 transcriptional terminator, positioned to blockreadthrough transcription resulting from DNA concatenation, but nottranscription resulting from repositioning of the permuted T7 promoterfrom the 5′ LTR to the 3′ LTR during reverse transcription.

2. Permuted Coding Region In a second embodiment, an indicator gene isrendered non-functional due to the relative location of the 5′ and 3′coding regions of the indicator gene, in that, the 31 coding regionprecedes rather than follows the 5′ coding region. This misplaced codingregion is referred to as a “permuted coding region.” The orientation ofthe non-functional indicator gene may be the same or opposite to that ofthe native or foreign promoter/enhancer of the viral vector, as mRNAcoding for a functional indicator gene will be produced in the event ofeither orientation. The non-functional indicator gene and its permutedcoding region is rendered functional by the action of one or more of thepatient-derived segment products. A second example of a non-functionalindicator gene with a permuted coding region in the case of HIV, placesa 5′ indicator gene coding region with an associated promoter in the 3′LTR U3 region and a 3′ indicator gene coding region in an upstreamlocation of the HIV genome, with each coding region having the sametranscriptional orientation as the viral LTRs. In both examples, the 5′and 3′ coding regions may also have associated splice donor and acceptorsequences, respectively, which may be heterologous or artificialsplicing signals. The indicator gene cannot be functionally transcribedeither by the associated promoter or viral promoters, as the permutedcoding region prevents the formation of functionally splicedtranscripts. The non-functional indicator gene in the resistance testvector is converted into a functional indicator gene by reversetranscriptase upon infection of the target cells, resulting from therepositioning of the 5′ and 3′ indicator gene coding regions relative toone another, by copying of the 3′ LTR to the 5′ LTR. Followingtranscription by the promoter associated with the 5′ coding region, RNAsplicing can join the 5′ and 3′ coding regions to produce a functionalindicator gene product. One example of a non-functional indicator genewith a permuted coding region in the case of HBV, places a 3′ indicatorgene coding region upstream or 5′ of the enhancer-promoter and the 5′coding region of the indicator gene. The transcriptional orientation ofthe indicator gene 5′ and 3′ coding regions are identical to oneanother, and the same as that of the indicator gene viral vector.However, as the indicator gene 5′ and 3′ coding regions are permuted inthe resistance test vectors (i.e., the 5′ coding region is downstream ofthe 3′ coding region), no mRNA is transcribed which can be spliced togenerate a functional indicator gene coding region. Following reversetranscription and circularization of the indicator gene viral vector,the indicator gene 3′ coding region is positioned downstream or 3′ tothe enhancer-promoter and 5′ coding regions thus permitting thetranscription of mRNA which can be spliced to generate a functionalindicator gene coding region.

3. Inverted Intron In a third embodiment, the indicator gene is renderednon-functional through use of an “inverted intron,” i.e. an introninserted into the coding sequence of the indicator gene with atranscriptional orientation opposite to that of the indicator gene. Theoverall transcriptional orientation of the indicator gene cassetteincluding its own, linked promoter, is opposite to that of the viralcontrol elements, while the orientation of the artificial intron is thesame as the viral control elements. Transcription of the indicator geneby its own linked promoter does not lead to the production of functionaltranscripts as the inverted intron cannot be spliced in thisorientation. Transcription of the indicator gene by the viral controlelements does, however, lead to the removal of the inverted intron byRNA splicing, although the indicator gene is still not functionallyexpressed as the resulting transcript has an antisense orientation.Following the reverse transcription of this transcript and integrationof the resultant retroviral DNA, or the circularization of hepadnavirusDNA, the indicator gene can be functionally transcribed using its ownlinked promoter as the inverted intron has been previously removed. Inthis case, the indicator gene itself may contain its own functionalpromoter with the entire transcriptional unit oriented opposite to theviral control elements. Thus the non-functional indicator gene is in thewrong orientation to be transcribed by the viral control elements and itcannot be functionally transcribed by its own promoter, as the invertedintron cannot be properly excised by splicing. However, in the case of aretrovirus and HIV specifically and hepadnaviruses, and HBVspecifically, transcription by the viral promoters (HIV LTR or HBVenhancer-promoter) results in the removal of the inverted intron bysplicing. As a consequence of reverse transcription of the resultingspliced transcript and the integration of the resulting provirus intothe host cell chromosome or circularization of the HBV vector, theindicator gene can now be functionally transcribed by its own promoter.The inverted intron, consisting of a splice donor and acceptor site toremove the intron, is preferably located in the coding region of theindicator gene in order to disrupt translation of the indicator gene.The splice donor and acceptor may be any splice donor and acceptor. Apreferred splice donor-receptor is the CMV IE splice donor and thesplice acceptor of the second exon of the human alpha globin gene(“intron A”).

Indicator Gene Viral Vector—Construction

As used herein, “indicator gene viral vector” refers to a vector(s)comprising an indicator gene and its control elements and one or moreviral genes. The indicator gene viral vector is assembled from anindicator gene cassette and a “viral vector,” defined below. Theindicator gene viral vector may additionally include an enhancer,splicing signals, polyadenylation sequences, transcriptionalterminators, or other regulatory sequences. Additionally the indicatorgene viral vector may be functional or nonfunctional. In the event thatthe viral segments which are the target of the anti-viral drug are notincluded in the indicator gene viral vector they are provided in asecond vector. An “indicator gene cassette” comprises an indicator geneand control elements. “Viral vector” refers to a vector comprising someor all of the following: viral genes encoding a gene product, controlsequences, viral packaging sequences, and in the case of a retrovirus,integration sequences. The viral vector may additionally include one ormore viral segments one or more of which may be the target of ananti-viral drug. Two examples of a viral vector which contain viralgenes are referred to herein as an “genomic viral vector” and a“subgenomic viral vector.” A “genomic viral vector” is a vector whichmay comprise a deletion of a one or more viral genes to render the virusreplication incompetent, but which otherwise preserves the mRNAexpression and processing characteristics of the complete virus. In oneembodiment for an HIV drug susceptibility and resistance test, thegenomic viral vector comprises the HIV gag-pol, vif, vpr, tat, rev, vpu,and nef genes (some, most or all of env may be deleted). A “subgenomicviral vector” refers to a vector comprising the coding region of one ormore viral genes which may encode the proteins that are the target(s) ofthe anti-viral drug. In the case of HIV, a preferred embodiment is asubgenomic viral vector comprising the HIV gag-pol gene. In the case ofHBV a preferred embodiment is a subgenomic viral vector comprising theHBV P gene. In the case of HIV, two examples of proviral clones used forviral vector construction are: HXB2 (Fisher et al., (1986) Nature, 320,367–371) and NL4-3, (Adachi et al., (1986) J. Virol., 59, 284–291). Inthe case of HBV, a large number of full length genomic sequences havebeen characterized and could be used for construction of HBV viralvectors: GenBank Nos. M54923, M38636, J02203 and X59795. The viralcoding genes may be under the control of a native enhancer/promoter or aforeign viral or cellular enhancer/promoter. A preferred embodiment foran HIV drug susceptibility and resistance test, is to place the genomicor subgenomic viral coding regions under the control of the nativeenhancer/promoter of the HIV-LTR U3 region or the CMV immediate-early(IE) enhancer/promoter. A preferred embodiment for an HBV drugsusceptibility and resistance test, is to place the genomic orsubgenomic viral coding regions under the control of the CMVimmediate-early (IE) enhancer/promoter. In the case of an indicator geneviral vector that contains one or more viral genes which are the targetsor encode proteins which are the targets of an anti-viral drug(s) thensaid vector contains the patient sequence acceptor sites. Thepatient-derived segments are inserted in the patient sequence acceptorsite in the indicator gene viral vector which is then referred to as theresistance test vector, as described above.

“Patient sequence acceptor sites” are sites in a vector for insertion ofpatient-derived segments and said sites may be: 1) unique restrictionsites introduced by site-directed mutagenesis into a vector; 2)naturally occurring unique restriction sites in the vector; or 3)selected sites into which a patient-derived segment may be insertedusing alternative cloning methods (e.g. UDG cloning). In one embodimentthe patient sequence acceptor site is introduced into the indicator geneviral vector. The patient sequence acceptor sites are preferably locatedwithin or near the coding region of the viral protein which is thetarget of the anti-viral drug. The viral sequences used for theintroduction of patient sequence acceptor sites are preferably chosen sothat no change, or a conservative change, is made in the amino acidcoding sequence found at that position. Preferably the patient sequenceacceptor sites are located within a relatively conserved region of theviral genome to facilitate introduction of the patient-derived segments.Alternatively, the patient sequence acceptor sites are located betweenfunctionally important genes or regulatory sequences. Patient-sequenceacceptor sites may be located at or near regions in the viral genomethat are relatively conserved to permit priming by the primer used tointroduce the corresponding restriction site into the patient-derivedsegment. To improve the representation of patient-derived segmentsfurther, such primers may be designed as degenerate pools to accommodateviral sequence heterogeneity, or may incorporate residues such asdeoxyinosine (I) which have multiple base-pairing capabilities. Sets ofresistance test vectors having patient sequence acceptor sites thatdefine the same or overlapping restriction site intervals may be usedtogether in the drug resistance and susceptibility tests to providerepresentation of patient-derived segments that contain internalrestriction sites identical to a given patient sequence acceptor site,and would thus be underrepresented in either resistance test vectoralone.

Host Cells

The resistance test vector is introduced into a host cell. Suitable hostcells are mammalian cells. Preferred host cells are derived from humantissues and cells which are the principle targets of viral infection. Inthe case of HIV these include human cells such as human T cells,monocytes, macrophage, dendritic cells, Langerhans cells, hematopoeiticstem cells or precursor cells, and other cells. In the case of HBV,suitable host cells include hepatoma cell lines (HepG2, Huh 7), primaryhuman hepatocytes, mammalian cells which can be-infected by pseudotypedHBV, and other cells. Human derived host cells will assure that theanti-viral drug will enter the cell efficiently and be converted by thecellular enzymatic machinery into the metabolically relevant form of theanti-viral inhibitor. Host cells are referred to herein as a “packaginghost cells,” “resistance test vector host cells,” or “target hostcells.” A “packaging host cell” refers to a host cell that provides thetrans-acting factors and viral packaging proteins required by thereplication defective viral vectors used herein, such as the resistancetest vectors, to produce resistance test vector viral particles. Thepackaging proteins may be provided for by the expression of viral genescontained within the resistance test vector itself, a packagingexpression vector(s), or both. A packaging host cell is a host cellwhich is transfected with one or more packaging expression vectors andwhen transfected with a resistance test vector is then referred toherein as a “resistance test vector host cell” and is sometimes referredto as a packaging host cell/resistance test vector host cell. Preferredhost cells for use as packaging host cells for HIV include 293 humanembryonic kidney cells (293, Graham, F. L. et al., J. Gen Virol. 36: 59,1977), BOSC23 (Pear et al., Proc. Natl. Acad. Sci. 90, 8392, 1993),tsa54 and tsa201 cell lines (Heinzel et al., J. Virol. 62, 3738, 1988),for HBV HepG2 (Galle and Theilmann, L. Arzheim.-Forschy Drug Res. (1990)40, 1380–1382). (Huh, Ueda, K et al. Virology *1989) 169, 213–216). A“target host cell” refers to a cell to be infected by resistance testvector viral particles produced by the resistance test vector host cellin which expression or inhibition of the indicator gene takes place.Preferred host cells for use as target host cells include human T cellleukemia cell lines including Jurkat (ATCC T1B–152), H9 (ATCC HTB-176),CEM (ATCC CCL-119), HUT78 (ATCC T1B–161), and derivatives thereof.

This invention is illustrated in the Experimental Detais section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

Experimental Details

General Materials and Methods

Most of the techniques used to construct vectors, and transfect andinfect cells, are widely practiced in the art, and most practitionersare familiar with the standard resource materials that describe specificconditions and procedures. However, for convenience, the followingparagraphs may serve as a guideline.

As used herein, “replication capacity” is defined herein is a measure ofhow well the virus replicates. This may also be referred to as viralfitness. In one embodiment, replication capacity can be measured byevaluating the ability of the virus to replicate in a single round ofreplication.

As used herein, “control resistance test vector” is defined as aresistance test vector comprising a standard viral sequence (forexample, HXB2, PNL4-3) and an indicator gene.

As used herein, “normalizing” is defined as standardizing the amount ofthe expression of indicator gene measured relative to the number ofviral particles giving rise to the expression of the indicator gene. Forexample, normalization is measured by dividing the amount of luciferaseactivity measured by the number of viral particles measured at the timeof infection.

“Plasmids” and “vectors” are designated by a lower case p followed byletters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

Construction of the vectors of the invention employs standard ligationand restriction techniques which are well understood in the art (seeAusubel et al., (1987) Current Protocols in Molecular Biology,Wiley—Interscience or Maniatis et al., (1992) in Molecular Cloning: Alaboratory Manual, Cold Spring Harbor Laboratory, N.Y.). Isolatedplasmids, DNA sequences, or synthesized oligonucleotides are cleaved,tailored, and religated in the form desired. The sequences of all DNAconstructs incorporating synthetic DNA were confirmed by DNA sequenceanalysis (Sanger et al. (1977) Proc. Natl. Acad. Sci. 74, 5463–5467).

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences, restrictionsites, in the DNA. The various restriction enzymes used herein arecommercially available and their reaction conditions, cofactors andother requirements are known to the ordinarily skilled artisan. Foranalytical purposes, typically 1 μg of plasmid or DNA fragment is usedwith about 2 units of enzyme in about 20 μl of buffer solution.Alternatively, an excess of restriction enzyme is used to insurecomplete digestion of the DNA substrate. Incubation times of about onehour to two hours at about 37° C. are workable, although variations canbe tolerated. After each incubation, protein is removed by extractionwith phenol/chloroform and the nucleic acid recovered from aqueousfractions by precipitation with ethanol. If desired, size separation ofthe cleaved fragments may be performed by polyacrylamide gel or agarosegel electrophoresis using standard techniques. A general description ofsize separations is found in Methods of Enzymology 65:499–560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° C. in 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mMMgCl₂, 6 mM DTT and 5–10 mM dNTPs. The Klenow fragment fills in at 5′sticky ends but chews back protruding 3′ single strands, even though thefour dNTPs are present. If desired, selective repair can be performed bysupplying only one of the dNTPs, or with selected dNTPs, within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with S1 nuclease orBal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 15–50 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 mg/ml BSA, 10 mM–50 mM NaCl, and either 40 μM ATP, 0.01–0.02(Weiss) units T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1 mMATP, 0.3–0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 33–100 μg/ml total DNA concentrations (5–100 mM total endconcentration). Intermolecular blunt end ligations (usually employing a10–30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

“Transient expression” refers to unamplified expression within about oneday to two weeks of transfection. The optimal time for transientexpression of a particular desired heterologous gene may vary dependingon several factors including, for example, any transacting factors whichmay be employed, translational control mechanisms and the host cell.Transient expression occurs when the particular plasmid that has beentransfected functions, i.e., is transcribed and translated. During thistime the plasmid DNA which has entered the cell is transferred to thenucleus. The DNA is in a nonintegrated state, free within the nucleus.Transcription of the plasmid taken up by the cell occurs during thisperiod. Following transfection the plasmid DNA may become degraded ordiluted by cell division. Random integration within the cell chromatinoccurs.

In general, vectors containing promoters and control sequences which arederived from species compatible with the host cell are used with theparticular host cell. Promoters suitable for use with prokaryotic hostsillustratively include the beta-lactamase and lactose promoter systems,alkaline phosphatase, the tryptophan (trp) promoter system and hybridpromoters such as tac promoter. However, other functional bacterialpromoters are suitable. In addition to prokaryotes, eukaryotic microbessuch as yeast cultures may also be used. Saccharomyces cerevisiae, orcommon baker's yeast is the most commonly used eukaryotic microorganism,although a number of other strains are commonly available. Promoterscontrolling transcription from vectors in mammalian host cells may beobtained from various sources, for example, the genomes of viruses suchas: polyoma, simian virus 40 (SV40), adenovirus, retroviruses, hepatitisB virus and preferably cytomegalovirus, or from heterologous mammalianpromoters, e.g. β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment thatalso contains the SV40 viral origin of replication. The immediate earlypromoter of the human cytomegalovirus is conveniently obtained as aHindIII E restriction fragment. Of course, promoters from the host cellor related species also are useful herein.

The vectors used herein may contain a selection gene, also termed aselectable marker. A selection gene encodes a protein, necessary for thesurvival or growth of a host cell transformed with the vector. Examplesof suitable selectable markers for mammalian cells include thedihydrofolate reductase gene (DHFR), the ornithine decarboxylase gene,the multi-drug resistance gene (mdr), the adenosine deaminase gene, andthe glutamine synthase gene. When such selectable markers aresuccessfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedia. The second category is referred to as dominant selection whichrefers to a selection scheme used in any cell type and does not requirethe use of a mutant cell line. These schemes typically use a drug toarrest growth of a host cell. Those cells which have a novel gene wouldexpress a protein conveying drug resistance and would survive theselection. Examples of such dominant selection use the drugs neomycin(Southern and Berg (1982) J. Molec. Appl. Genet. 1, 327), mycophenolicacid (Mulligan and Berg (1980) Science 209, 1422), or hygromycin (Sugdenet al. (1985) Mol. Cell. Biol. 5, 410–413). The three examples givenabove employ bacterial genes under eukaryotic control to conveyresistance to the appropriate drug neomycin (G418 or genticin), xgpt(mycophenolic acid) or hygromycin, respectively.

“Transfection” means introducing DNA into a host cell so that the DNA isexpressed, whether functionally expressed or otherwise; the DNA may alsoreplicate either as an extrachromosomal element or by chromosomalintegration. Unless otherwise provided, the method used herein fortransfection of the host cells is the calcium phosphate co-precipitationmethod of Graham and van der Eb (1973) Virology 52, 456–457. Alternativemethods for transfection are electroporation, the DEAE-dextran method,lipofection and biolistics (Kriegler (1990) Gene Transfer andExpression: A Laboratory Manual, Stockton Press).

Host cells may be transfected with the expression vectors of the presentinvention and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants oramplifying genes. Host cells are cultured in F12:DMEM (Gibco) 50:50 withadded glutamine. The culture conditions, such as temperature, pH and thelike, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The following examples merely illustrate the best mode now known forpracticing the invention, but should not be construed to limit theinvention. All publications and patent applications cited in thisspecification are herein incorporated by reference in their entirety asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference.

EXAMPLE 1 Phenotypic Drug Susceptibility and Resistance Test UsingResistance Test Vectors

Phenotypic drug susceptibility and resistance tests are carried outusing the means and methods described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319) which is herebyincorporated by reference.

In these experiments patient-derived segment(s) corresponding to the HIVprotease and reverse transcriptase coding regions were eitherpatient-derived segments amplified by the reversetranscription-polymerase chain reaction method (RT-PCR) using viral RNAisolated from viral particles present in the serum of HIV-infectedindividuals or were mutants of wild type HIV-1 made by site directedmutagenesis of a parental clone of resistance test vector DNA. Isolationof viral RNA was performed using standard procedures (e.g. RNAgentsTotal RNA Isolation System, Promega, Madison Wis. or RNAzol, Tel-Test,Friendswood, Tex.). The RT-PCR protocol was divided into two steps. Aretroviral reverse transcriptase [e.g. Moloney MuLV reversetranscriptase (Roche Molecular Systems, Inc., Branchburg, N.J.), oravian myeloblastosis virus (AMV) reverse transcriptase, (BoehringerMannheim, Indianapolis, Ind.)] was used to copy viral RNA into cDNA. ThecDNA was then amplified using a thermostable DNA polymerase [e.g. Taq(Roche Molecular Systems, Inc., Branchburg, N.J.), Tth (Roche MolecularSystems, Inc., Branchburg, N.J.), PrimeZyme (isolated from Thermusbrockianus, Biometra, Gottingen, Germany)] or a combination ofthermostable polymerases as described for the performance of “long PCR”(Barnes, W. M., (1994) Proc. Natl. Acad. Sci, USA 91, 2216–2220) [e.g.Expand High Fidelity PCR System (Taq+Pwo), (Boehringer Mannheim.Indianapolis, Ind.) OR GeneAmp XL PCR kit (Tth+Vent), (Roche MolecularSystems, Inc., Branchburg, N.J.)].

PCR6 (Table 5, #1) is used for reverse transcription of viral RNA intocDNA. The primers, ApaI primer (PDSApa, Table 5, #2) and AgeI primer(PDSAge, Table 5, #3) used to amplify the “test” patient-derivedsegments contained sequences resulting in ApaI and AgeI recognitionsites being introduced into both ends of the PCR product, respectively.

Resistance test vectors incorporating the “test” patient-derivedsegments were constructed as described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319) (see FIG. 1) using anamplified DNA product of 1.5 kB prepared by RT-PCR using viral RNA as atemplate and oligonucleotides PCR6 (#1), PDSApa (#2) and PDSAge (#3) asprimers, followed by digestion with ApaI and AgeI or the isoschizomerPinA1. To ensure that the plasmid DNA corresponding to the resultantresistance test vector comprises a representative sample of the HIVviral quasi-species present in the serum of a given patient, many (>100)independent E. coli transformants obtained in the construction of agiven resistance test vector were pooled and used for the preparation ofplasmid DNA.

A packaging expression vector encoding an amphotrophic MuLV 4070A envgene product enables production in a resistance test vector host cell ofresistance test vector viral particles which can efficiently infecthuman target cells. Resistance test vectors encoding all HIV genes withthe exception of env were used to transfect a packaging host cell (oncetransfected the host cell is referred to as a resistance test vectorhost cell). The packaging expression vector which encodes theamphotrophic MuLV 4070A env gene product is used with the resistancetest vector to enable production in the resistance test vector host cellof infectious pseudotyped resistance test vector viral particles.

Resistance tests performed with resistance test vectors were carried outusing packaging host and target host cells consisting of the humanembryonic kidney cell line 293 (Cell Culture Facility, UC San Francisco,SF, Calif.) or the Jurkat leukemic T-cell line (Arthur Weiss, UC SanFrancisco, SF, Calif.).

Resistance tests were carried out with resistance test vectors using twohost cell types. Resistance test vector viral particles were produced bya first host cell (the resistance test vector host cell) that wasprepared by transfecting a packaging host cell with the resistance testvector and the packaging expression vector. The resistance test vectorviral particles were then used to infect a second host cell (the targethost cell) in which the expression of the indicator gene is measured(see FIG. 2).

The resistance test vectors containing a functional luciferase genecassette were constructed and host cells were transfected with theresistance test vector DNA. The resistant test vectors containedpatient-derived reverse transcriptase and protease DNA sequences thatencode proteins which were either susceptible or resistant to theantiretroviral agents, such as nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors and proteaseinhibitors. The resistance test vector viral particles produced bytransfecting the resistance test vector DNA into host cells, either inthe presence or absence of protease inhibitors, were used to infecttarget host cells grown either in the absence of NRTI or NNRTI or in thepresence of increasing concentrations of the drug. Luciferase activityin infected target host cells in the presence of drug was compared tothe luciferase activity in infected target host cells in the absence ofdrug. Drug resistance was measured as the concentration of drug requiredto inhibit by 50% the luciferase activity detected in the absence ofdrug (inhibitory concentration 50%, IC50). The IC50 values weredetermined by plotting percent drug inhibition vs. log₁₀ drugconcentration.

Host cells were seeded in 10-cm-diameter dishes and were transfected oneday after plating with resistance test vector plasmid DNA and theenvelope expression vector. Transfections were performed using acalcium-phosphate co-precipitation procedure. The cell culture mediacontaining the DNA precipitate was replaced with fresh medium, from oneto 24 hours, after transfection. Cell culture media containingresistance test vector viral particles was harvested one to four daysafter transfection and was passed through a 0.45-mm filter before beingstored at −80° C. HIV capsid protein (p24) levels in the harvested cellculture media were determined by an EIA method as described by themanufacturer (SIAC; Frederick, Md.). Before infection, target cells (293and 293/T) were plated in cell culture media. Control infections wereperformed using cell culture media from mock transfections (no DNA) ortransfections containing the resistance test vector plasmid DNA withoutthe envelope expression plasmid. One to three or more days afterinfection the media was removed and cell lysis buffer (Promega) wasadded to each well. Cell lysates were assayed for luciferase activity.The inhibitory effect of the drug was determined using the followingequation:% luciferase inhibition=[1−(RLUluc[drug]RLUluc)]×100where RLUluc [drug] is the relative light unit of luciferase activity ininfected cells in the presence of drug and RLUluc is the Relative LightUnit of luciferase activity in infected cells in the absence of drug.IC50 values were obtained from the sigmoidal curves that were generatedfrom the data by plotting the percent inhibition of luciferase activityvs. the log₁₀ drug concentration. Examples of drug inhibition curves areshown in (FIG. 3).

EXAMPLE 2 An In Vitro Assay Using Resistance Test Vectors and SiteDirected Mutants to Correlate Phenotypes and Genotypes Associated withHIV Drug Susceptibility and Resistance

Phenotypic susceptibility analysis of patient HIV samples Resistancetest vectors are constructed as described in example 1. Resistance testvectors, or clones derived from the resistance test vector pools, aretested in a phenotypic assay to determine accurately and quantitativelythe level of susceptibility to a panel of anti-retroviral drugs. Thispanel of anti-retroviral drugs may comprise members of the classes knownas nucleoside-analog reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), and proteaseinhibitors (PRIs). The panel of drugs can be expanded as new drugs ornew drug targets become available. An IC50 is determined for eachresistance test vector pool for each drug tested. The pattern ofsusceptibility to all of the drugs tested is examined and compared toknown patterns of susceptibility.

A patient sample can be further examined for genotypic changescorrelated with the pattern of susceptibility observed.

Genotypic Analysis of Patient HIV Samples

Resistance test vector DNAs, either pools or clones, are analyzed by anyof the genotyping methods described in Example 1. In one embodiment ofthe invention, patient HIV sample sequences are determined using viralRNA purification, RT/PCR and ABI chain terminator automated sequencing.The sequence that is determined is compared to control sequences presentin the database or is compared to a sample from the patient prior toinitiation of therapy, if available. The genotype is examined forsequences that are different from the control or pre-treatment sequenceand correlated to the observed phenotype.

Phenotypic Susceptibility Analysis of Site Directed Mutants

Genotypic changes that are observed to correlate with changes inphenotypic patterns of drug susceptibility are evaluated by constructionof resistance test vectors containing the specific mutation on adefined, wild-type (drug susceptible) genetic background. Mutations maybe incorporated alone and/or in combination with other mutations thatare thought to modulate the susceptibility of HIV to a certain drug orclass of drugs. Mutations are introduced into the resistance test vectorthrough any of the widely known methods for site-directed mutagenesis.In one embodiment of this invention the mega-primer PCR method forsite-directed mutagenesis is used. A resistance test vector containingthe specific mutation or group of mutations are then tested using thephenotypic susceptibility assay described above and the susceptibilityprofile is compared to that of a genetically defined wild-type (drugsusceptible) resistance test vector which lacks the specific mutations.Observed changes in the pattern of phenotypic susceptibility to theantiretroviral drugs tested are attributed to the specific mutationsintroduced into the resistance test vector.

EXAMPLE 3

Using Resistance Test Vectors to Correlate Genotypes and PhenotypesAssociated with Changes in PRI Drug Susceptibility in HIV.

Phenotypic Analysis of Patient 0732

A resistance test vector was constructed as described in example 1 froma patient sample designated as 0732. This patient had been previouslytreated with nelfinavir. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1–313 of RT. The patient derived segment wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-0732. RTV-0732 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-0732 inwhich there was a decrease in both nelfinavir and indinavirsusceptibility (increased resistance) and an increase in amprenavirsusceptibility (see FIG. 4 and Table 1). Patient sample 0732 wasexamined further for genotypic changes associated with the pattern ofsusceptibility.

Determination of Genotype of Patient 0732

RTV-0732 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions K14R, I15V,K20T, E35D, M36I, R41K, I62V, L63Q and N88S. K14R, I15V, E35D, R41K andI62V are naturally occurring polymorphisms in HIV-1 PR and are notassociated with reduced susceptibility to any drug. M36I has previouslybeen described to be associated with resistance to ritonavir andnelfinavir (Shihazi, 1998). N88S has previously been described to beassociated with resistance to nelfinavir (Patick A A C, 42: 2637 (1998)and an investigational PRI, SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 627

A resistance test vector was constructed as described in example 1 froma patient sample designated as 627. This patient had been treated withindinavir. Isolation of viral RNA and RT/PCR was used to generate apatient derived segment that comprised viral sequences coding for all ofPR and aa 1–313 of RT. The patient derived segment was inserted into anindicator gene viral vector to generate a resistance test vectordesignated RTV-627. RTV-627 was tested using a phenotypic susceptibilityassay to determine accurately and quantitatively the level ofsusceptibility to a panel of anti-retroviral drugs. This panel ofanti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-627 inwhich there was a decrease in indinavir and nelfinavir susceptibility(increased resistance) and an increase in amprenavir and saquinavirsusceptibility. Patient sample 627 was examined further for genotypicchanges associated with the pattern of susceptibility.

Determination of Genotype of Patient 627

RTV-627 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions 13I/V, E35D,M46L, L63P, I64V, I73V and N88S. I13V, E35D and I64V are naturallyoccurring polymorphisms in HIV-1 PR and are not associated with reducedsusceptibility to any drug. M46L has previously been described to beassociated with resistance to indinavir and amprenavir. L63P haspreviously been described to be associated with resistance to indinavirand nelfinavir. N88S has previously been described to be associated withresistance to nelfinavir (Patick, 1998) and an investigational PRI,SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 1208

A resistance test vector was constructed as described in example 1 froma patient sample designated as 1208. This patient had been previouslytreated with nelfinavir. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1–313 of RT. The patient derived segment wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-1208. RTV-1208 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-1208 inwhich there was a decrease in indinavir and nelfinavir susceptibility(increased resistance) and an increase in amprenavir susceptibility.Patient sample 1208 was examined further for genotypic changesassociated with the pattern of susceptibility.

Determination of Genotype of Patient 1208

RTV-1208 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions I62V, L63P,V77I, and N88S. I62V is a naturally occurring polymorphism in HIV-1 PRand is not associated with reduced susceptibility to any drug. L63P haspreviously been described to be associated with resistance to indinavirand nelfinavir. V77I has previously been described to be associated withresistance to nelfinavir. N88S has previously been described to beassociated with resistance to nelfinavir (Patick, 1998) and aninvestigational PRI, SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 360

A resistance test vector was constructed as described in example 1 froma patient sample designated as 360. This patient had been previouslytreated with indinavir. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1–313 of RT. The patient derived segment wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-360. RTV-360 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-360 inwhich there was a decrease in indinavir and nelfinavir susceptibility(increased resistance) and an increase in amprenavir susceptibility.Patient sample 360 was examined further for genotypic changes associatedwith the pattern of susceptibility.

Determination of Genotype of Patient 360

RTV-360 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions I13V, K20M,M36V, N37A, M46I, I62V, L63P, N88S, and I93L. I13V, N37A and I62V arenaturally occurring polymorphisms in HIV-1 PR and are not associatedwith reduced susceptibility to any drug. K20M has previously beendescribed to be associated with resistance to indinavir. M46I haspreviously been described to be associated with resistance to indinavir,ritonavir, nelfinavir and amprenavir. L63P has previously been describedto be associated with resistance to indinavir and nelfinavir. N88S haspreviously been described to be associated with resistance to nelfinavir(Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 0910

A resistance test vector was constructed as described in example 1 froma patient sample designated as 0910. This patient had been previouslytreated with nelfinavir. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1–313 of RT. The patient derived segment wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-0910. RTV-0910 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-0910 inwhich there was a decrease in indinavir and nelfinavir susceptibility(increased resistance) and an increase in amprenavir susceptibility.Patient sample 0910 was examined further for genotypic changesassociated with the pattern of susceptibility.

Determination of Genotype of Patient 0910

RTV-0910 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions M46I, L63P,V77I, N88S and I93I/L. I13V, K14R, N37D and I193L are naturally occuringpolymorphism in HIV-1 PR and is not associated with reducedsusceptibility to any drug. V77I has previously been described to beassociated with resistance to nelfinavir. M46I has previously beendescribed to be associated with resistance to indinavir, ritonavir,nelfinavir and amprenavir. L63P has previously been described to beassociated with resistance to indinavir and nelfinavir. N88S haspreviously been described to be associated with resistance to nelfinavir(Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 3542

A resistance test vector was constructed as described in example 1 froma patient sample designated as 3542. This patient had been treated withindinavir. Isolation of viral RNA and RT/PCR was used to generate apatient derived segment that comprised viral sequences coding for all ofPR and aa 1–313 of RT. The patient derived segment was inserted into anindicator gene viral vector to generate a resistance test vectordesignated RTV-3542. RTV-3542 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-3542 inwhich there was a decrease in indinavir, nelfinavir and ritonavirsusceptibility (increased resistance) and an increase in amprenavirsusceptibility. Patient sample 3542 was examined further for genotypicchanges associated with the pattern of susceptibility.

Determination of Genotype of Patient 3542

RTV-3542 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions I13V, K14R,N37D, M46I, L63P, N88S and I93L. K14R and N37A/D are naturally occurringpolymorphisms in HIV-1 PR and are not associated with reducedsusceptibility to any drug. M46I has previously been described to beassociated with resistance to indinavir, ritonavir, nelfinavir andamprenavir. L63P has previously been described to be associated withresistance to indinavir and nelfinavir. N88S has previously beendescribed to be associated with resistance to nelfinavir (Patick, 1998)and an investigational PRI, SC55389A (Smidt, 1997).

Phenotypic Analysis of Patient 3654

A resistance test vector was constructed as described in example 1 froma patient sample designated as 3654. This patient had been previouslytreated with ritonavir. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1–313 of RT. The patient derived segment wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-3654. RTV-3654 was tested using a phenotypicsusceptibility assay to determine accurately and quantitatively thelevel of susceptibility to a panel of anti-retroviral drugs. This panelof anti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI, ddC, and abacavir), NNRTIs (delavirdine, nevirapineand efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavirand amprenavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the PRIs was observed for patient sample RTV-3654 inwhich there was a decrease in indinavir and nelfinavir susceptibility(increased resistance) and an increase in amprenavir susceptibility.Patient sample 3654 was examined further for genotypic changesassociated with the pattern of susceptibility.

Determination of Genotype of Patient 3654

RTV-3654 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. PR mutations were noted at positions I13V, R41K,M46I, L63P, V77I, N88S and I93L. I13V, R41K and I93L are naturallyoccurring polymorphism in HIV-1 PR and is not associated with reducedsusceptibility to any drug. M46I has previously been described to beassociated with resistance to indinavir, ritonavir, nelfinavir andamprenavir. L63P has previously been described to be associated withresistance to indinavir and nelfinavir. V77I has previously beendescribed to be associated with resistance to nelfinavir. N88S haspreviously been described to be associated with resistance to aninvestigational PRI, SC55389A (Smidt, 1997).

EXAMPLE 4 Using Site Directed Mutants to Correlate Genotypes andPhenotypes Associated with Changes in PRI Drug Susceptibility in HIV

Site Directed Mutagenesis

Resistance test vectors were constructed containing the N88S mutationalone and in combination with other substitutions in PR (L63P, V77I andM46L) known to modulate the HIV susceptibility to PRIs. Mutations wereintroduced into the resistance test vector using the mega-primer PCRmethod for site-directed mutagenesis. (Sakar G and Sommar S S (1994)Biotechniques 8(4), 404–407) First, a resistance test vector wasconstructed that harbors a unique RsrII restriction site 590 bpdownstream of the ApaI restriction site. The 590 bp ApaI-RsrII fragmentthus contains the entire protease region. This site was introduced bysite-specific oligonucleotide-directed mutagenesis using primer #4. Allsubsequent mutants were constructed by fragment-exchange of thewild-type ApaI-RsrII fragment in the parent vector with the equivalentfragment carrying the respective mutations.

A resistance test vector containing the N88S mutation (N88S-RTV) wastested using the phenotypic susceptibility assay described above and theresults were compared to that of a genetically defined resistance testvector that was wild type at position 88. The pattern of phenotypicsusceptibility to the PRIs in the N88S-RTV was altered as compared towild type. In the context of an otherwise wild type background (i.e.N88S mutation alone) the N88S-RTV was more susceptible to bothamprenavir and ritonavir and slightly less susceptible to nelfinavircompared to the wild type control RTV (see Table 2).

A resistance test vector containing the N88S mutation along with theL63P mutation (L63P-N88S-RTV) was tested using the phenotypicsusceptibility assay described above and the results were compared tothat of a genetically defined resistance test vector that was wild typeat positions 63 and 88. The L63P-N88S-RTV showed decreasedsusceptibility to both indinavir and nelfinavir and an increase in thesusceptibility to amprenavir compared the wild-type control RTV (seeTable 2). Thus it appears that the introduction of a second mutation,L63P, in addition to N88S, results in a reduction in susceptibility tonelfinavir and indinavir while the increased susceptibility toamprenavir is maintained.

A resistance test vector containing the N88S mutation along with theL63P mutation and the V77I mutation (L63P-V77I-N88S-RTV) was testedusing the phenotypic susceptibility assay described above and theresults were compared to that of a genetically defined resistance testvector that was wild type at positions 63 and 77 and 88. The RTVcontaining mutations at these positions, L63P-V77I-N88S-RTV, showed adecrease in susceptibility to both indinavir and nelfinavir and anincrease in the susceptibility to amprenavir compared to the wild-typecontrol RTV (see FIG. 5 and Table 2). Thus it appears that theintroduction of a third mutation, V77I, in addition to L63P and N88S,results in a reduction in susceptibility to nelfinavir and indinavirwhile the increased susceptibility to amprenavir is maintained.

The N88S mutation was also introduced into an RTV containing additionalmutations at positions L63P and M46L (M46L+L63P+N88S). The RTVcontaining mutations at these positions, M46L-L63P-N88S-RTV showed adecrease in susceptibility to nelfinavir and a slight decrease insusceptibility to indinavir and an increase in the susceptibility toamprenavir compared to the wild-type control RTV (see FIG. 5 and Table2) Thus it appears that the introduction of a third mutation, M46L, inaddition to L63P and N88S, results in a reduction in susceptibility tonelfinavir and indinavir while the increased susceptibility toamprenavir is maintained.

A resistance test vector containing the N88S mutation along with theM46L mutation, the L63P mutation, and the V77I mutation(M46L-L63P-V77I-N88S-RTV) was tested using the phenotypic susceptibilityassay described above and the results were compared to that of agenetically defined resistance test vector that was wild type atpositions 46, 63, 77 and 88. The RTV containing mutations at these fourpositions, M46L-L63P-V77I-N88S-RTV showed a decrease in susceptibilityto nelfinavir and indinavir and an increase in the susceptibility toamprenavir compared to the wild-type control RTV (see FIG. 5 and Table2). Thus it appears that the introduction of a fourth mutation, V77I, inaddition to L63P, M46L and N88S results in a reduction in susceptibilityto nelfinavir and indinavir while the increased susceptibility toamprenavir is maintained. A resistance test vector containing the L63Pmutation (L63P-RTV) was tested using the phenotypic susceptibility assaydescribed above and the results were compared to that of a geneticallydefined resistance test vector that was wild type at position 63. Thepattern of phenotypic susceptibility to the PRIs in the L63P-RTV wassimilar to wild type with no significant changes in susceptibility tothe PRIs observed.

The L63P mutation was also introduced into an RTV containing anadditional mutation at position V77I. The L63P-V77I-RTV showed a slightdecrease in susceptibility to nelfinavir compared to the wild-typecontrol RTV (see FIG. 5 and Table 2).

EXAMPLE 5 Predicting Response to Protease Inhibitors by Characterizationof Amino Acid 88 of HIV-1 Protease

In one embodiment of this invention, changes in the amino acid atposition 88 of the protease protein of HIV-1 is evaluated using thefollowing method comprising: (i) collecting a biological sample from anHIV-1 infected subject; (ii) evaluating whether the biological samplecontains nucleic acid encoding HIV-1 protease having an asparagine toserine mutation at codon 88 (N88S); and (iii) determining susceptibilityto protease inhibitors (PRI).

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 88 ofthe HIV-1 protease is mutated to serine, can be performed using variousmethods, such as direct characterization of the viral nucleic acidencoding protease or direct characterization of the protease proteinitself. Defining the amino acid at position 88 of protease can beperformed by direct characterization of the protease protein byconventional or novel amino acid sequencing methodologies, epitoperecognition by antibodies or other specific binding proteins orcompounds. Alternatively, the amino acid at position 88 of the HIV-1protease protein can be defined by characterizing amplified copies ofHIV-1 nucleic acid encoding the protease protein. Amplification of theHIV-1 nucleic acid can be performed using a variety of methodologiesincluding reverse transcription-polymerase chain reaction (RT-PCR),NASBA, SDA, RCR, or 3SR. The nucleic acid sequence encoding HIV proteaseat codon 88 can be determined by direct nucleic acid sequencing usingvarious primer extension-chain termination (Sanger, ABI/PE and VisibleGenetics) or chain cleavage (Maxam and Gilbert) methodologies or morerecently developed sequencing methods such as matrix assisted laserdesorption-ionization time of flight (MALDI-TOF) or mass spectrometry(Sequenom, Gene Trace Systems). Alternatively, the nucleic acid sequenceencoding amino acid position 88 can be evaluated using a variety ofprobe hybridization methodologies, such as genechip hybridizationsequencing (Affymetrix), line probe assay (LiPA; Murex), anddifferential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of proteaseinhibitor susceptibility and of whether amino acid position 88 of HIV-1protease was wild type or serine was carried out using a phenotypicsusceptibility assay or genotypic assay, respectively, using resistancetest vector DNA prepared from the biological sample. In one embodiment,the plasma sample was collected, viral RNA was purified and an RT-PCRmethodology was used to amplify a patient derived segment encoding theHIV-1 protease and reverse transcriptase regions. The amplified patientderived segments were then incorporated, via DNA ligation and bacterialtransformation, into an indicator gene viral vector thereby generating aresistance test vector. Resistance test vector DNA was isolated from thebacterial culture and the phenotypic susceptibility assay was carriedout as described in Example 1. The results of the phenotypicsusceptibility assay with a patient sample having an N88S mutation in PRis shown in FIG. 4. The nucleic acid (DNA) sequence of the patientderived HIV-1 protease and reverse transcriptase regions from patientsample 0732 was determined using a fluorescence detection chaintermination cycle sequencing methodology (ABI/PE). The method was usedto determine a consensus nucleic acid sequence representing thecombination of sequences of the mixture of HIV-1 variants existing inthe subject sample (representing the quasispecies), and to determine thenucleic acid sequences of individual variants.

Phenotypic and Genotypic Correlation of Mutations at Amino Acid 88 ofHIV-1 Protease.

Phenotypic susceptibility profiles of patient samples and site directedmutants showed that amprenavir susceptibility correlated with thepresence of the N88S mutation in HIV-1 protease. Phenotypicsusceptibility profiles of patient samples and site directed mutantsshowed that a significant increase in amprenavir susceptibility(decreased resistance) correlated with a mutation in the nucleic acidsequence encoding the amino acid serine (S) at position 88 of HIV-1protease.

Phenotypic susceptibility profiles of patient samples and site directedmutants showed reduction in amprenavir susceptibility (decreasedresistance) and a decrease in susceptibility to nelfinavir and indinavirwith the amino acid serine at position 88 when the PR mutations atpositions 63, 77 or 46 were also present (L63P, V77I, or M46L).

EXAMPLE 6 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes Associated with Alterations in PRI Susceptibilitywith Viral Fitness

Luciferase activity measured in the absence of drug for the sevenresistance test vectors constructed from the patient viruses containingthe N88S PR mutation ranged from 0.7 to 16% of control (Table 3).Although these viruses also contain multiple mutations in reversetranscriptase, which could also contribute to a reduction in viralfitness, the data suggest that viruses containing the N88S mutation areless fit than wild type. To confirm this observation, the luciferaseexpression level for the site-directed mutant resistance test vectorswas also examined.

Viruses containing N88S as the only substitution produced only 1.0% ofthe luciferase activity in the absence of drug (Table 4). This reductionwas substantially alleviated by the addition of the L63P substitution(20.7%) or by addition of the combinations of L63P/V77I (29.3%) orM46L/L63P (28.0%). The L63P or L63P/V77I mutants had equivalent orincreased relative luciferase activity compared to wild type (163.9 and75.6%, respectively).

When the K20T substitution was added to the N88S background, eitheralone or in combination with L63P, only background levels of luciferaseactivity was detected. Sequence analysis confirmed the absence ofadditional mutations, which might render the vector inactive. Thus thecombination of the K20T and N88S substitutions correlates with a severedefect in fitness.

EXAMPLE 7 Predicting Response to Protease Inhibitors by Characterizationof Amino Acid 82 of HIV-1 Protease

In one embodiment of this invention, changes in the amino acid atposition 82 of the protease protein of HIV-1 are evaluated using thefollowing method comprising: (i) collecting a biological sample from anHIV-1 infected subject; (ii) evaluating whether the biological samplecontains nucleic acid encoding HIV-1 protease having a valine to alanine(V82A), phenylalanine (V82F), serine (V82S), or threonine (V82T)substitution at codon 82; and (iii) determining susceptibility toprotease inhibitors (PRI).

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 82 ofthe HIV-1 protease is mutated to alanine, phenylalanine, serine, orthreonine, can be performed using various methods, such as directcharacterization of the viral nucleic acid encoding protease or directcharacterization of the protease protein itself. Defining the amino acidat position 82 of protease can be performed by direct characterizationof the protease protein by conventional or novel amino acid sequencingmethodologies, epitope recognition by antibodies or other specificbinding proteins or compounds. Alternatively, the amino acid at position82 of the HIV-1 protease protein can be defined by characterizingamplified copies of HIV-1 nucleic acid encoding the protease protein.Amplification of the HIV-1 nucleic acid can be performed using a varietyof methodologies including reverse transcription-polymerase chainreaction (RT-PCR), NASBA, SDA, RCR, or 3SR. The nucleic acid sequenceencoding HIV protease at codon 82 can be determined by direct nucleicacid sequencing using various primer extension-chain termination(Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam andGilbert) methodologies or more recently developed sequencing methodssuch as matrix assisted laser desorption-ionization time of flight(MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems).Alternatively, the nucleic acid sequence encoding amino acid position 82can be evaluated using a variety of probe hybridization methodologies,such as genechip hybridization sequencing (Affymetrix), line probe assay(LiPA; Murex), and differential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of proteaseinhibitor susceptibility and of whether amino acid position 82 of HIV-1protease was wild type or alanine, phenylalanine, serine, or threonine,was carried out using a phenotypic susceptibility assay or genotypicassay, respectively, using resistance test vector DNA prepared from thebiological sample. In one embodiment, the plasma sample was collected,viral RNA was purified and an RT-PCR methodology was used to amplify apatient derived segment encoding the HIV-1 protease and reversetranscriptase regions. The amplified patient derived segments were thenincorporated, via DNA ligation and bacterial transformation, into anindicator gene viral vector thereby generating a resistance test vector.Resistance test vector DNA was isolated from the bacterial culture andthe phenotypic susceptibility assay was carried out and analyzed asdescribed in Example 1.

The nucleic acid (DNA) sequence of the patient derived HIV-1 proteaseand reverse transcriptase regions was determined using a fluorescencedetection chain termination cycle sequencing methodology (ABI/PE). Themethod was used to determine a consensus nucleic acid sequencerepresenting the combination of sequences of the mixture of HIV-1variants existing in the subject sample (representing the quasispecies),and to determine the nucleic acid sequences of individual variants.Genotypes are analyzed as lists of amino acid differences between virusin the patient sample and a reference laboratory strain of HIV-1, NL4-3.Genotypes and corresponding phenotypes (fold-change in IC50 values) areentered in a relational database linking these two results with patientinformation. Large datasets can then be assembled from patient virussamples sharing particular characteristics, such as the presence of anygiven mutation, or combination of mutations or reduced susceptibility toany drug or combination of drugs.

(a) Protease Inhibitor Susceptibility of Viruses Containing Mutations atAmino Acid 82 of HIV-1 Protease.

Phenotypic susceptibility profiles of 75 patient virus samples whichcontained a mutation at position 82 (V82A, F, S, or T), but no otherprimary mutations, were analyzed. According to most publishedguidelines, such viruses are expected to be resistant to ritonavir,nelfinavir, indinavir, and saquinavir. However, 8%, 20%, 23%, and 73% ofthese samples were phenotypically susceptible to these four proteaseinhibitors, respectively (see Table 6). Thus, particularly for indinavirand saquinavir, there was poor correlation between the presence ofmutations at position 82 and drug susceptibility.

(b) Indinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 82 and One Secondary Mutation in HIV-1 Protease.

Indinavir resistance in viruses containing mutations at position 82 wasevaluated with respect to the presence of other specific mutations.Decreased indinavir susceptibility (fold-change in IC₅₀ greater than2.5) in viruses containing V82A, F, S, or T but no other primarymutations was correlated with the presence of mutations at secondarypositions. Reduced indinavir susceptibility was observed in 20 samplescontaining mutations at both positions 24 and 82 (100%) and in 27samples with both 71 and 82 (100%) (See Table 7). The combination ofmutations at position 82 with mutations at other positions (e.g. 54, 46,10, and 63) also significantly increased the proportion of samples thathad reduced indinavir susceptibility (Table 7).

(c) Saquinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 82 and One Secondary Mutation in HIV-1 Protease.

Saquinavir resistance in viruses containing mutations at position 82 wasevaluated with respect to the presence of other specific mutations.Decreased saquinavir susceptibility (fold-change in IC₅₀ greater than2.5) in viruses containing V82A, F, S, or T but no other primarymutations was correlated with the presence of mutations at secondarypositions. Reduced saquinavir susceptibility was observed in 4 of 5samples containing mutations at both positions 20 and 82 (80%) and in 8of 11 samples with both 36 and 82 (73%) (See Table 8). The combinationof mutations at position 82 with mutations at other positions (e.g. 24,71, 54, and 10) also significantly increased the proportion of samplesthat had reduced saquinavir susceptibility (Table 8).

(d) Indinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 82 and Many Secondary Mutations in HIV-1Protease.

Indinavir resistance in viruses containing mutations at position 82 wasevaluated with respect to the presence of a defined number of othermutations. Decreased indinavir susceptibility (fold-change in IC₅₀greater than 2.5) in viruses containing V82A, F, S, or T but no otherprimary mutations was correlated with the number of mutations atsecondary positions. Reduced indinavir susceptibility was observed in100% of samples with V82A, F, S, or T and at least 6 other secondarymutations (See Table 9). The proportion of samples that had reducedindinavir susceptibility increased significantly in samples with V82A,F, S, or T combined with 3 to 5 other secondary mutations (Table 9).

(e) Saquinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 82 and Many Secondary Mutations in HIV-1Protease.

Saquinavir resistance in viruses containing mutations at position 82 wasevaluated with respect to the presence of a defined number of othermutations. Decreased saquinavir susceptibility (fold-change in IC₅₀greater than 2.5) in viruses containing V82A, F, S, or T but no otherprimary mutations was correlated with the number of mutations atsecondary positions. Reduced saquinavir susceptibility was observed in60 to 76% of samples with V82A, F, S, or T and at least 5 othersecondary mutations (See Table 9). The proportion of samples that hadreduced saquinivir susceptibility increased significantly in sampleswith V82A, F, S, or T combined with 3 or 4 other secondary mutations(Table 9).

EXAMPLE 8 Predicting Response to Protease Inhibitors by Characterizationof Amino Acid 90 of HIV-1 Protease

In one embodiment of this invention, changes in the amino acid atposition 90 of the protease protein of HIV-1 are evaluated using thefollowing method comprising: (i) collecting a biological sample from anHIV-1 infected subject; (ii) evaluating whether the biological samplecontains nucleic acid encoding HIV-1 protease having a leucine tomethionine (L90M) substitution at codon 90; and (iii) determiningsusceptibility to protease inhibitors (PRI).

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 90 ofthe HIV-1 protease is mutated to methionine, can be performed usingvarious methods, such as direct characterization of the viral nucleicacid encoding protease or direct characterization of the proteaseprotein itself. Defining the amino acid at position 90 of protease canbe performed by direct characterization of the protease protein byconventional or novel amino acid sequencing methodologies, epitoperecognition by antibodies or other specific binding proteins orcompounds. Alternatively, the amino acid at position 90 of the HIV-1protease protein can be defined by characterizing amplified copies ofHIV-1 nucleic acid encoding the protease protein. Amplification of theHIV-1 nucleic acid can be performed using a variety of methodologiesincluding reverse transcription-polymerase chain reaction (RT-PCR),NASBA, SDA, RCR, or 3SR. The nucleic acid sequence encoding HIV proteaseat codon 90 can be determined by direct nucleic acid sequencing usingvarious primer extension-chain termination (Sanger, ABI/PE and VisibleGenetics) or chain cleavage (Maxam and Gilbert) methodologies or morerecently developed sequencing methods such as matrix assisted laserdesorption-ionization time of flight (MALDI-TOF) or mass spectrometry(Sequenom, Gene Trace Systems). Alternatively, the nucleic acid sequenceencoding amino acid position 90 can be evaluated using a variety ofprobe hybridization methodologies, such as genechip hybridizationsequencing (Affymetrix), line probe assay (LIPA; Murex), anddifferential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of proteaseinhibitor susceptibility and of whether amino acid position 90 of HIV-1protease was wild type or methionine, was carried out using a phenotypicsusceptibility assay or genotypic assay, respectively, using resistancetest vector DNA prepared from the biological sample. In one embodiment,the plasma sample was collected, viral RNA was purified and an RT-PCRmethodology was used to amplify a patient derived segment encoding theHIV-1 protease and reverse transcriptase regions. The amplified patientderived segments were then incorporated, via DNA ligation and bacterialtransformation, into an indicator gene viral vector thereby generating aresistance test vector. Resistance test vector DNA was isolated from thebacterial culture and the phenotypic susceptibility assay was carriedout and analyzed as described in Example 1.

The nucleic acid (DNA) sequence of the patient derived HIV-1 proteaseand reverse transcriptase regions was determined using a fluorescencedetection chain termination cycle sequencing methodology (ABI/PE). Themethod was used to determine a consensus nucleic acid sequencerepresenting the combination of sequences of the mixture of HIV-1variants existing in the subject sample (representing the quasispecies),and to determine the nucleic acid sequences of individual variants.Genotypes are analyzed as lists of amino acid differences between virusin the patient sample and a reference laboratory strain of HIV-1, NL4-3.Genotypes and corresponding phenotypes (fold-change in IC50 values) areentered in a relational database linking these two results with patientinformation. Large datasets can then be assembled from patient virussamples sharing particular characteristics, such as the presence of anygiven mutation, or combination of mutants, or reduced susceptibility toany drug or combination of drugs.

(a) Protease Inhibitor Susceptibility of Viruses Containing Mutations atAmino Acid 90 of HIV-1 Protease.

Phenotypic susceptibility profiles of 58 patient virus samples whichcontained a mutation at position 90 (L90M), but no other primarymutations, were analyzed. According to most published guidelines, suchviruses are expected to be resistant to ritonavir, nelfinavir,indinavir, and saquinavir. However, 28%, 9%, 31%, and 47% of thesesamples were phenotypically susceptible to these four proteaseinhibitors, respectively (see Table 6). Thus, particularly for indinavirand saquinavir, there was poor correlation between the presence ofmutations at position 90 and drug susceptibility.

(b) Indinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 90 and One Secondary Mutation in HIV-1 Protease.

Indinavir resistance in viruses containing mutations at position 90 wasevaluated with respect to the presence of other specific mutations.Decreased indinavir susceptibility (fold-change in IC₅₀ greater than2.5) in viruses containing L90M but no other primary mutations wascorrelated with the presence of mutations at secondary positions.Reduced indinavir susceptibility was observed in 17 of 19 samplescontaining mutations at both positions 73 and 90 (89%) and in 16 of 18samples with both 71 and 90 (89%) (See Table 10). The combination ofmutations at position 90 with mutation at position 46 also significantlyincreased the proportion of samples that had reduced indinavirsusceptibility (Table 10).

(c) Saquinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 90 and One Secondary Mutation in HIV-1 Protease.

Saquinavir resistance in viruses containing mutations at position 90 wasevaluated with respect to the presence of other specific mutations.Decreased saquinavir susceptibility (fold-change in IC₅₀ greater than2.5) in viruses containing L90M but no other primary mutations wascorrelated with the presence of mutations at secondary positions.Reduced saquinavir susceptibility was observed in 15 of 19 samplescontaining mutations at both positions 73 and 90 (79%) and in 14 of 18samples with both 71 and 90 (78%) (See Table 11). The combination ofmutations at position 90 with mutations at other positions (e.g. 77 and10) also significantly increased the proportion of samples that hadreduced saquinavir susceptibility (Table 1).

(d) Indinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 90 and Many Secondary Mutations in HIV-1Protease.

Indinavir resistance in viruses containing mutations at position 90 wasevaluated with respect to the presence of a defined number of othermutations. Decreased indinavir susceptibility (fold-change in IC₅₀greater than 2.5) in viruses containing L90M but no other primarymutations was correlated with the number of mutations at secondarypositions. Reduced indinavir susceptibility was observed in 100% ofsamples with L90M and at least 5 other secondary mutations had (SeeTable 12). The proportion of samples that had reduced indinavirsusceptibility increased significantly in samples with L90M combinedwith 3 or 4 other secondary mutations (Table 12).

(e) Saquinavir Susceptibility of Viruses Containing Combinations ofMutations at Amino Acid 90 and Many Secondary Mutations in HIV-1Protease.

Saquinavir resistance in viruses containing mutations at position 90 wasevaluated with respect to the presence of a defined number of othermutations. Decreased saquinavir susceptibility (fold-change in IC₅₀greater than 2.5) in viruses containing L90M but no other primarymutations was correlated with the number of mutations at secondarypositions. Reduced saquinavir susceptibility was observed in 100% ofsamples with L90M and at least 5 other secondary mutations (See Table12). The proportion of samples that had reduced saquinivirsusceptibility increased significantly in samples with L90M combinedwith 3 or 4 other secondary mutations (Table 12).

EXAMPLE 9 Predicting Response to Protease Inhibitors by Characterizationof Amino Acids 82 and 90 of HIV-1 Protease

In one embodiment of this invention, changes in the amino acid atposition 82 and 90 of the protease protein of HIV-1 are evaluated usingthe following method comprising: (i) collecting a biological sample froman HIV-1 infected subject; (ii) evaluating whether the biological samplecontains nucleic acid encoding HIV-1 protease having a valine to alanine(V82A), phenylalanine (V82F), serine (V82S), or threonine (V82T)substitution at codon 82 or a leucine to methionine at position 90(L90M); and (iii) determining susceptibility to protease inhibitors(PRI).

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 82 ofthe HIV-1 protease is mutated to alanine, phenylalanine, serine, orthreonine or at position 90 to methionine, can be performed usingvarious methods, such as direct characterization of the viral nucleicacid encoding protease or direct characterization of the proteaseprotein itself. Defining the amino acid at positions 82 and 90 ofprotease can be performed by direct characterization of the proteaseprotein by conventional or novel amino acid sequencing methodologies,epitope recognition by antibodies or other specific binding proteins orcompounds. Alternatively, the amino acid at positions 82 and 90 of theHIV-1 protease protein can be defined by characterizing amplified copiesof HIV-1 nucleic acid encoding the protease protein. Amplification ofthe HIV-1 nucleic acid can be performed using a variety of methodologiesincluding reverse transcription-polymerase chain reaction (RT-PCR),NASBA, SDA, RCR, or 3SR. The nucleic acid sequence encoding HIV proteaseat codons 82 and 90 can be determined by direct nucleic acid sequencingusing various primer extension-chain termination (Sanger, ABI/PE andVisible Genetics) or chain cleavage (Maxam and Gilbert) methodologies ormore recently developed sequencing methods such as matrix assisted laserdesorption-ionization time of flight (MALDI-TOF) or mass spectrometry(Sequenom, Gene Trace Systems). Alternatively, the nucleic acid sequenceencoding amino acid positions 82 and 90 can be evaluated using a varietyof probe hybridization methodologies, such as genechip hybridizationsequencing (Affymetrix), line probe assay (LiPA; Murex), anddifferential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of proteaseinhibitor susceptibility and of whether amino acid positions 82 and 90of HIV-1 protease was wild type or alanine, phenylalanine, serine, orthreonine in the case of position 82 and methionine at position 90, wascarried out using a phenotypic susceptibility assay or genotypic assay,respectively, using resistance test vector DNA prepared from thebiological sample. In one embodiment, plasma sample was collected, viralRNA was purified and an RT-PCR methodology was used to amplify a patientderived segment encoding the HIV-1 protease and reverse transcriptaseregions. The amplified patient derived segments were then incorporated,via DNA ligation and bacterial transformation, into an indicator geneviral vector thereby generating a resistance test vector. Resistancetest vector DNA was isolated from the bacterial culture and thephenotypic susceptibility assay was carried out and analyzed asdescribed in Example 1.

The nucleic acid (DNA) sequence of the patient derived HIV-1 proteaseand reverse transcriptase regions was determined using a fluorescencedetection chain termination cycle sequencing methodology (ABI/PE). Themethod was used to determine a consensus nucleic acid sequencerepresenting the combination of sequences of the mixture of HIV-1variants existing in the subject sample (representing the quasispecies),and to determine the nucleic acid sequences of individual variants.Genotypes are analyzed as lists of amino acid differences between virusin the patient sample and a reference laboratory strain of HIV-1, NL4-3.Genotypes and corresponding phenotypes (fold-change in IC50 values) areentered in a relational database linking these two results with patientinformation. Large datasets can then be assembled from patient virussamples sharing particular characteristics, such as the presence of anygiven mutation or reduced susceptibility to any drug or combination ofdrugs.

Protease Inhibitor Susceptibility of Viruses Containing Mutations atAmino Acids 82 and 90 of HIV-1 Protease.

Phenotypic susceptibility profiles of 33 patient virus samples whichcontained mutations at positions 82 (V82A, F, S, or T) and 90 (L90M),but no other primary mutations, were analyzed. According to mostpublished guidelines, such viruses are expected to be resistant toritonavir, nelfinavir, indinavir, and saquinavir. However, 9% and 21% ofthese samples were phenotypically susceptible to indinavir andsaquinavir, respectively (see Table 6). Thus, particularly forsaquinavir, there was poor correlation between the presence of mutationsat positions 82 and 90 and drug susceptibility.

EXAMPLE 10

Measuring Replication Fitness Using Resistance Test Vectors

A means and method is provided for accurately measuring and reproducingthe replication fitness of HIV-1. This method for measuring replicationfitness is applicable to other viruses, including, but not limited tohepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitisC virus) and herpesviruses (human cytomegalovirus). This example furtherprovides a means and method for measuring the replication fitness ofHIV-1 that exhibits reduced drug susceptibility to reverse transcriptaseinhibitors and protease inhibitors. This method can be used formeasuring replication fitness for other classes of inhibitors of HIV-1replication, including, but not limited to integration, virus assembly,and virus attachment and entry.

Replication fitness tests are carried out using the means and methodsfor phenotypic drug susceptibility and resistance tests described inU.S. Pat. No. 5,837,464 (International Publication Number WO 97/27319)which is hereby incorporated by reference.

In these experiments patient-derived segment(s) corresponding to the HIVprotease and reverse transcriptase coding regions were eitherpatient-derived segments amplified by the reversetranscription-polymerase chain reaction method (RT-PCR) using viral RNAisolated from viral particles present in the serum of HIV-infectedindividuals or were mutants of wild type HIV-1 made by site directedmutagenesis of a parental clone of resistance test vector DNA.Resistance test vectors are also referred to as “fitness test vectors”when used to evaluate replication fitness. Isolation of viral RNA wasperformed using standard procedures (e.g. RNAgents Total RNA IsolationSystem, Promega, Madison Wis. or RNAzol, Tel-Test, Friendswood, Tex.).The RT-PCR protocol was divided into two steps. A retroviral reversetranscriptase [e.g. Moloney MuLV reverse transcriptase (Roche MolecularSystems, Inc., Branchburg, N.J.), or avian myeloblastosis virus (AMV)reverse transcriptase, (Boehringer Mannheim, Indianapolis, Ind.)] wasused to copy viral RNA into cDNA. The cDNA was then amplified using athermostable DNA polymerase [e.g. Taq (Roche Molecular Systems, Inc.,Branchburg, N.J.), Tth (Roche Molecular Systems, Inc., Branchburg,N.J.), PrimeZyme (isolated from Thermus brockianus, Biometra, Gottingen,Germany)] or a combination of thermostable polymerases as described forthe performance of “long PCR” (Barnes, W. M., (1994) Proc. Natl. Acad.Sci, USA 91, 2216–2220) [e.g. Expand High Fidelity PCR System (Taq+Pwo),(Boehringer Mannheim. Indianapolis, Ind.) OR GeneAmp XL PCR kit(Tth+Vent), (Roche Molecular Systems, Inc., Branchburg, N.J.)].

PCR6 (Table 5, #1) is used for reverse transcription of viral RNA intocDNA. The primers, ApaI primer (PDSApa, Table 5, #2) and AgeI primer(PDSAge, Table 5, #3) used to amplify the “test” patient-derivedsegments contained sequences resulting in ApaI and AgeI recognitionsites being introduced into both ends of the PCR product, respectively.

Fitness test vectors incorporating the “test” patient-derived segmentswere constructed as described in U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319) (see FIG. 1) using an amplified DNAproduct of 1.5 kB prepared by RT-PCR using viral RNA as a template andoligonucleotides PCR6 (#1), PDSApa (#2) and PDSAge (#3) as primers,followed by digestion with ApaI and AgeI or the isoschizomer PinA1. Toensure that the plasmid DNA corresponding to the resultant fitness testvector comprises a representative sample of the HIV viral quasi-speciespresent in the serum of a given patient, many (>100) independent E. colitransformants obtained in the construction of a given fitness testvector were pooled and used for the preparation of plasmid DNA.

A packaging expression vector encoding an amphotrophic MuLV 4070A envgene product enables production in a fitness test vector host cell offitness test vector viral particles which can efficiently infect humantarget cells. Fitness test vectors encoding all HIV genes with theexception of env were used to transfect a packaging host cell (oncetransfected the host cell is referred to as a fitness test vector hostcell). The packaging expression vector which encodes the amphotrophicMuLV 4070A env gene product is used with the resistance test vector toenable production in the fitness test vector host cell of infectiouspseudotyped fitness test vector viral particles.

Fitness tests performed with fitness test vectors were carried out usingpackaging host and target host cells consisting of the human embryonickidney cell line 293 (Cell Culture Facility, UC San Francisco, SF,Calif.)

Fitness tests were carried out with fitness test vectors using two hostcell types. Fitness test vector viral particles were produced by a firsthost cell (the fitness test vector host cell) that was prepared bytransfecting a packaging host cell with the fitness test vector and thepackaging expression vector. The fitness test vector viral particleswere then used to infect a second host cell (the target host cell) inwhich the expression of the indicator gene is measured (see FIG. 6).

The fitness test vectors containing a functional luciferase genecassette were constructed and host cells were transfected with thefitness test vector DNA. The fitness test vectors containedpatient-derived reverse transcriptase and protease DNA sequences thatencode proteins which were either susceptible or resistant to theantiretroviral agents, such as nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors and proteaseinhibitors.

The amount of luciferase activity detected in the infected cells is usedas a direct measure of “infectivity”, “replication capacity” or“fitness”, i.e. the ability of the virus to complete a single round ofreplication. Relative fitness is assessed by comparing the amount ofluciferase activity produced by patient derived viruses to the amount ofluciferase activity produced by a well-characterized reference virus(wildtype) derived from a molecular clone of HIV-1, for example NL4-3 orHXB2. Fitness measurements are expressed as a percent of the reference,for example 25%, 50%, 75%, 100% or 125% of reference (FIGS. 6B, 6C).

Host cells were seeded in 10-cm-diameter dishes and were transfected oneday after plating with fitness test vector plasmid DNA and the envelopeexpression vector. Transfections were performed using acalcium-phosphate co-precipitation procedure. The cell culture mediacontaining the DNA precipitate was replaced with fresh medium, from oneto 24 hours, after transfection. Cell culture media containing fitnesstest vector viral particles was harvested one to four days aftertransfection and was passed through a 0.45-mm filter before being storedat −80° C. HIV capsid protein (p24) levels in the harvested cell culturemedia were determined by an EIA method as described by the manufacturer(SIAC; Frederick, Md.). Before infection, target cells (293 and 293/T)were plated in cell culture media. Control infections were performedusing cell culture media from mock transfections (no DNA) ortransfections containing the fitness test vector plasmid DNA without theenvelope expression plasmid. One to three or more days after infectionthe media was removed and cell lysis buffer (Promega) was added to eachwell. Cell lysates were assayed for luciferase activity. Alternatively,cells were lysed and luciferase was measured by adding Steady-Glo(Promega) reagent directly to each well without aspirating the culturemedia from the well.

EXAMPLE 11 Measuring Replication Fitness of Viruses with Deficiencies inReverse Transcriptase Activity

A means and method is provided for identifying mutations in reversetranscriptase that alter replication fitness. A means and method isprovided for identifying mutations that alter replication fitness andcan be used to identify mutations associated with other aspects of HIV-1replication, including, but not limited to integration, virus assembly,and virus attachment and entry. This example also provides a means andmethod for quantifying the affect that specific mutations reversetranscriptase have on replication fitness. A means and method forquantifying the affect that specfic protease and reverse transcriptasemutations have on replication fitness to mutations in other viral genesinvolved in HIV-1 replication, including, but not limited to the gag,pol, and envelope genes is also provided.

Fitness test vectors were constructed as described in example 10.Fitness test vectors derived from patient samples or clones derived fromthe fitness test vector pools, or fitness test vectors were engineeredby site directed mutagenesis to contain specific mutations, and weretested in a fitness assay to determine accurately and quantitatively therelative fitness compared to a well-characterized reference standard. Apatient sample was examined for increased or decreased reversetranscriptase activity and correlated with the relative fitness observed(FIG. 8).

Reverse Transcriptase Activity of Patient HIV Samples

Reverse transcriptase activity can be measured by any number of widelyused assay procedures, including but not limited to homopolymericextension using (e.g. oligo dT:poly rC) or real time PCR based onmolecular beacons (reference Kramer) or 5′ exonuclease activity (Lie andPetropoulos, 1996). In one embodiment, virion associated reversetranscriptase activity was measured using a quantitative PCR assay thatdetects the 5′ exonuclease activity associated with thermo-stable DNApolymerases (FIG. 8). In one embodiment of the invention, the fitness ofthe patient virus was compared to a reference virus to determine therelative fitness compared to “wildtype” viruses that have not beenexposed to reverse transcriptase inhibitor drugs. In another embodiment,the fitness of the patient virus was compared to viruses collected fromthe same patient at different timepoints, for example prior toinitiating therapy, before or after changes in drug treatment, or beforeor after changes in virologic (RNA copy number), immunologic (CD4T-cells), or clinical (opportunistic infection) markers of diseaseprogression.

Genotypic Analysis of Patient HIV Samples

Fitness test vector DNAs, either pools or clones, are analyzed by any ofthe genotyping methods described in Example 1. In one embodiment of theinvention, patient HIV sample sequences were determined using viral RNApurification, RT/PCR and ABI chain terminator automated sequencing. Thesequence was determined and compared to reference sequences present inthe database or compared to a sample from the patient prior toinitiation of therapy. The genotype was examined for sequences that aredifferent from the reference or pre-treatment sequence and correlated tothe observed fitness.

Fitness Analysis of Site Directed Mutants

Genotypic changes that are observed to correlate with changes in fitnesswere evaluated by construction of fitness vectors containing thespecific mutation on a defined, wild-type (drug susceptible) geneticbackground. Mutations may be incorporated alone and/or in combinationwith other mutations that are thought to modulate the fitness of avirus. Mutations were introduced into the fitness test vector throughany of the widely known methods for site-directed mutagenesis. In oneembodiment of this invention the mega-primer PCR method forsite-directed mutagenesis is used. A fitness test vector containing thespecific mutation or group of mutations were then tested using thefitness assay described in Example 10 and the fitness was compared tothat of a genetically defined wild-type (drug susceptible) fitness testvector which lacks the specific mutations. Observed changes in fitnessare attributed to the specific mutations introduced into the resistancetest vector. In several related embodiments of the invention, fitnesstest vectors containing site directed mutations in reverse transcriptasethat result in amino acid substitutions at position 190 (G190A, G190S,G190C, G190E, G190V, G190T) and that display different amounts ofreverse transcriptase activity were constructed and tested for fitness(FIG. 9). The fitness results were correlated with specific reversetranscriptase amino acid substituions and fitness.

EXAMPLE 12 Measuring Replication Fitness of Viruses with Deficiencies inProtease Activity

A means and method for identifying mutations in protease that alterreplication fitness is provided.

This example provides the means and methods for identifying mutationsthat alter replication fitness for various components of HIV-1replication, including, but not limited to integration, virus assembly,and virus attachment and entry. This example also provides a means andmethod for quantifying the affect that specific mutations in protease orreverse transcriptase have on replication fitness. This method can beused for quantifying the effect that specific protease mutations have onreplication fitness and can be used to quantify the effect of othermutations in other viral genes involved in HIV-1 replication, including,but not limited to the gag, pol, and envelope genes.

Fitness test vectors were constructed as described in example 10.Fitness test vectors derived from patient samples or clones derived fromthe fitness test vector pools, or fitness test vectors engineered bysite directed mutagenesis to contain specific mutations, were tested ina fitness assay to determine accurately and quantitatively the relativefitness compared to a well-characterized reference standard. A patientsample was examined further for increased or decreased protease activitycorrelated with the relative fitness observed (FIG. 8).

Protease Activity of Patient HIV Samples

Protease activity can be measured by any number of widely used assayprocedures, including but not limited to in vitro reactions that measureprotease cleavage activity (reference Erickson). In one embodiment,protease cleavage of the gag polyprotein (p55) was measured by Westernblot analyis using an anti-capsid (p24) antibody (FIG. 8). In oneembodiment of the invention, the fitness of the patient virus wascompared to a reference virus to determine the relative fitness comparedto “wildtype” viruses that have not been exposed to protease inhibitordrugs. In another embodiment, the fitness of the patient virus wascompared to viruses collected from the same patient at differenttimepoints, for example prior to initiating therapy, before or afterchanges in drug treatment, or before or after changes in virologic (RNAcopy number), immunologic (CD4 T-cells), or clinical (opportunisticinfection) markers of disease progression.

Genotypic Analysis of Patient HIV Samples

Fitness test vector DNAs, either pools or clones, are analyzed by any ofthe genotyping methods described in Example 1. In one embodiment of theinvention, patient HIV sample sequences were determined using viral RNApurification, RT/PCR and ABI chain terminator automated sequencing. Thesequence was determined and compared to reference sequences present inthe database or compared to a sample from the patient prior toinitiation of therapy, if available. The genotype was examined forsequences that are different from the reference or pre-treatmentsequence and correlated to the observed fitness.

Fitness Analysis of Site Directed Mutants

Genotypic changes that are observed to correlate with changes in fitnessare evaluated by construction of fitness vectors containing the specificmutation on a defined, wild-type (drug susceptible) genetic background.Mutations may be incorporated alone and/or in combination with othermutations that are thought to modulate the fitness of a virus. Mutationsare introduced into the fitness test vector through any of the widelyknown methods for site-directed mutagenesis. In one embodiment of thisinvention the mega-primer PCR method for site-directed mutagenesis isused. A fitness test vector containing the specific mutation or group ofmutations are then tested using the fitness assay described in Example10 and the fitness is compared to that of a genetically definedwild-type (drug susceptible) fitness test vector which lacks thespecific mutations. Observed changes in fitness are attributed to thespecific mutations introduced into the fitness test vector. In severalrelated embodiments of the invention, fitness test vectors containingsite directed mutations in reverse protease that result in amino acidsubstitutions at positions 30, 63, 77, 90 (list from FIG. 10) and thatdisplay different amounts of protease activity are constructed andtested for fitness (FIG. 10). The fitness results enable the correlationbetween specific protease amino acid substitutions and changes in viralfitness.

EXAMPLE 13 Measuring Replication Fitness and Drug Susceptibility in aLarge Patient Population

This example describes the high incidence of patient samples withreduced replication fitness. This example also describes the generalcorrelation between reduced drug susceptibility and reduced replicationfitness. This example further describes the occurrence of viruses withreduced fitness in patients receiving protease inhibitor and/or reversetranscriptase inhibitor treatment. This example further describes theincidence of patient samples with reduced replication fitness in whichthe reduction in fitness is due to altered protease processing of thegag polyprotein (p55). This example further describes the incidence ofprotease mutations in patient samples that exhibit low, moderate ornormal (wildtype) replication fitness. This example further describesprotease mutations that are frequently observed, either alone or incombination, in viruses that exhibit reduced replication capacity. Thisexample also describes the incidence of patient samples with reducedreplication fitness in which the reduction in fitness is due to alteredreverse transcriptase activity. This example describes the occurrence ofviruses with reduced replication fitness in patients failingantiretroviral drug treatment.

Fitness/resistance test vectors were constructed as described in example10. Fitness and drug susceptibility was measured in 134 random patientsamples that were received for routing phenotypic testing by theVirologic Clinical Reference Laboratory. Fitness assays were performedas described in Example 10. Drug susceptibility testing and genotypingof the protease region was performed as described in Example 1. Reversetranscriptase activity was measured as described in Example 11. Proteaseprocessing was measured as described in Example 12.

Drug Susceptibility of Patient Viruses

Reduced drug susceptibility was observed for a majority of the patientvirus samples (Table A). 66 percent of the viruses exhibited large(define as >10X of the reference) reductions in susceptibility to one ormore NRTI drugs. 52 percent of the viruses exhibited large reductions insusceptibility to one or more NNRTI drugs. 45 percent of the virusesexhibited large reductions in susceptibility to one or more PRI drugs.

Fitness of Patient Viruses

Reduced replication fitness was observed for a majority of the patientvirus samples (Table A). Forty one percent of the viruses exhibitedlarge reductions in replication fitness (<25% of the reference). Another45% had moderate reductions (between 25–75% of the reference) inreplication fitness. A minority of the patient samples (14%) displayedreplication fitness that approached or exceeded “wildtype” levels (>75%of the reference). Viruses with reduced drug susceptibility, were muchmore likely to display reduced replication fitness (FIGS. 11,12,13, and14).

Protease Mutations in Patient Viruses

Greater than 10 mutations in protease were observed in a majority of thepatient virus samples (Table A). Viruses with reduced fitness were muchmore likely to contain 10 or more protease mutations (FIG. 14). Sixtytwo percent of the viruses that exhibited large reductions inreplication fitness (<25% of the reference) contained 10 or moreprotease mutations. Twenty two percent of the viruses with moderatereductions (between 25–75% of the reference) in fitness contained 10 ormore protease mutations. Only 5% of the viruses that displayedreplication fitness that approached or exceeded “wildtype” levels (>75%of the reference) contained 10 or more protease mutations (Table A).Certain protease mutations either alone (D30N) or in combination (L90Mplus K20T, or M46I, or 73, or N88D) were observed at high incidences inviruses with reduced fitness (FIGS. 14 and 15).

Protease Processing of Patient Viruses

Reduced protease processing of the p55 gag polyprotein was observed in amajority of the patient virus samples (Table A). Viruses with reducedfitness were much more likely to display reduced protease processing;defined as having detectable amounts of the p41 intermediate cleavageproduct (FIGS. 11,14 and 16). Seventy one percent of the viruses thatexhibited large reductions in replication fitness (<25% of thereference) displayed reduced protease processing. Eighteen percent ofthe viruses with moderate fitness reductions (between 25–75% of thereference) displayed reduced protease processing. Only 10% of theviruses that displayed replication fitness that approached or exceeded“wildtype” levels (>75% of the reference) exhibited reduced proteaseprocessing (Table A). Certain protease mutations (D30N, M46I/L, G48V,I54L/A/S/T/V, and I84V) were observed at high incidences in viruses withreduced protease processing of the p55 gag polyprotein (FIG. 17).

Reverse Transcriptase of Patient Viruses

Reduced reverse transcriptase activity processing was observed in aminority of the patient virus samples (Table A). Viruses with reducedfitness were much more likely to display reduced reverse transcriptaseactivity. Fourteen percent of the viruses that exhibited largereductions in replication fitness (<25% of the reference) displayedreduced reverse transcriptase activity. Only 2% of the viruses withmoderate fitness reductions (between 25–75% of the reference) displayedreduced reverse transcriptase activity. None of the viruses thatdisplayed replication fitness that approached or exceeded “wildtype”levels (>75% of the reference) exhibited reduced reverse transcriptaseactivity.

EXAMPLE 14 Measuring Replication Fitness to Guide Treatment Decisions

A means and method for using replication fitness measurements to guidethe treatment of HIV-1 is provided. This example further provides ameans and method for using replication fitness measurements to guide thetreatment of patients failing antiretroviral drug treatment. Thisexample further provides the means and methods for using replicationfitness measurements to guide the treatment of patients newly infectedwith HIV-1.

Guiding treatment of patients with multi-drug resistant virus:Fitness/resistance test vectors were constructed as described in example10. Fitness and drug susceptibility were measured on serial longitudinalsamples collected weekly for 12 weeks from 18 patients. These patientswere considered failing a protease inhibitor (typically indinavir)containing regimen and had incomplete suppression of virus replicationbased on routine viral load testing (>2,500 copies/mL). Phenotypic drugsusceptibility testing indicated that these patient viruses weremulti-drug resistant. Each patient agreed to interrupt therapy for aperiod of at least 12 weeks. Phenotypic drug susceptibility assays wereperformed as described in Example 1 on serial samples collected justprior to interrupting therapy and weekly during the period ofinterruption. Fitness assays were performed as described in Example 10on serial samples collected just prior to interrupting therapy andweekly during the period of interruption. Protease processing wasmeasured as described in Example 12.

Of the 18 patients that interrupted therapy, 16 patients had resistantviruses that regained susceptibility to antiretroviral drugs during theperiod of treatment interruption. The phenotypic test results of arepresentative patient are shown in FIG. 18. Typically, susceptibilityreturned to all drug classes simultaneously, consistent with there-emergence of a minor population of drug sensitive virus. In therepresentative example shown in Figure M, drug sensitivity was abruptlyrestored between weeks 9 and 10. Genotypic analysis (DNA sequence ofprotease and reverse transcriptase) are also consistent with there-emergence of a drug sensitive virus. These data show the loss of mostor all drug resistance mutation simultaneously (data not shown). Thedata are not consistent with random back mutations. Back mutations wouldpredict that restored susceptibility to drugs would occur unevenly fordifferent drug classes and/or within a drugs within the same class.

Generally, the re-emergence of the drug susceptible virus was alsoaccompanied by a simultaneous increase in replication fitness. Thisrelationship is clearly evident for the representative virus (FIG. 19).Several other examples with less frequent timepoints are shown in FIG.O. Virus from patients that did not revert to drug susceptibility afterinterruption generally did not exhibit an increase in replicationfitness, nor did viruses from patients that did not interrupt treatment(FIG. 20). The data indicate that the drug sensitive virus thatre-emerged after treatment interruption is able to replicate better thanthe drug resistant virus that was present before treatment wasinterrupted. The re-emergence of drug susceptible virus in this group ofpatients was also accompanied by an increase in viral load and adecrease in DC4 T-cells, indicators of disease progression. Thus,fitness information can be used to guide treatment of patients thatharbor multi-drug resistant virus and are considering treatmentinterruption. If the patient virus is drug resistant but has lowreplication capacity, the patient and the physician should considercontinuing drug treatment to prevent the re-emergence of a drugsensitive virus with higher replication capacity and greaterpathogenecity. Alternatively, if the patient virus is drug resistant andhas high replication capacity, the patient and the physician mayconsider interrupting treatment to spare the patient from the harmfuland unpleasant side effects of antiretroviral drugs that are notproviding clinical benefit.

Furthermore, physicians may choose to perform routine replicationfitness assays for patients that have multi-drug resistant virus. Thisassay could be used to monitor the replication fitness of patientviruses when complete suppression of virus replication is not possibledue to multi-drug resistance. The assay would be used to guide treatmentdecisions that prevent the drug resistant virus with low replicationfitness from increasing its replication fitness. In this way, physiciansmay prolong the usefulness of antiretroviral drugs despite the presenceof drug resistant virus in the patient.

Guiding Treatment of Newly Infected Patients:

Patients that maintain high virus loads (setpoint) after acute infectionare more likely to exhibit accelerated disease progression. Therefore,it is advantageous for this class of patient to initiate antiretroviraldrug treatment as soon as possible after diagnosis with HIV-1 infection.In conjunction with viral load, fitness measurements of viruses in newlyinfected patients may provide a useful measurement to identify thoseindividuals that will develop elevated setpoints after primary infectionand consequently are likely to exhibit accelerated disease progression.Fitness measurements may guide the decision to treat immediately afterdiagnosis or a some later time point.

EXAMPLE 15 Measuring Saquinavir Susceptibility of Viruses ContainingVarious Amino Acid Substitutions in Protease at Position 82

This example provides a means and method for identifying mutations inprotease that affect susceptibility (increased or decreased) tosaquinavir.

In one embodiment of this invention, the effects of combination ofmutations at position 82 (for example, V82A, V82F, V82S, or V82T areevaluated using the following method comprising: (i) collecting abiological sample from an HIV-1 infected subject; (ii) evaluatingwhether the HIV-1 in the sample contains nucleic acid encoding proteasehaving a valine to alanine (V82A), phenylalanine (V82F), serine (V82S),or threonine (V82T) substitution at position 82 or a leucine tomethionine substitution at position 90 (L90M); and (iii) determiningsusceptibility to protease inhibitors (PRIs).

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 82 ofthe HIV-1 protease is mutated to alanine, phenylalanine, or threonine,can be performed using various methods, such as direct characterizationof the viral nucleic acid encoding protease or direct characterizationof the protease protein itself. Defining the amino acid at position 82of protease can be performed by direct characterization of the proteaseprotein by conventional or novel amino acid sequencing methodologies,epitope recognition by antibodies or other specific binding proteins orcompounds. Alternatively, the amino acid at position 82 of the HIV-1protease protein can be defined by characterizing amplified copies ofHIV-1 nucleic acid encoding the protease protein. Amplification of theHIV-1 nucleic acid can be performed using a variety of methodologiesincluding reverse transcription-polymerase chain reaction (RT-PCR),NASBA, SDA, RCR, or 3SR. The nucleic acid sequence encoding HIV proteaseat codon 82 can be determined by direct nucleic acid sequencing usingvarious primer extension-chain termination (Sanger, ABI/PE and VisibleGenetics) or chain cleavage (Maxam and Gilbert) methodologies or morerecently developed sequencing methods such as matrix assisted laserdesorption-ionization time of flight (MALDI-TOF) or mass spectrometry(Sequenom, Gene Trace Systems). Alternatively, the nucleic acid sequenceencoding amino acid position 82 can be evaluated using a variety ofprobe hybridization methodologies, such as genechip hybridizationsequencing (Affymetrix), line probe assay (LIPA; Murex), anddifferential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of the effectsof mutations at amino acid position 82 of HIV-1 protease on proteaseinhibitor susceptibility, was carried out using a phenotypicsusceptibility assay using resistance test vector DNA prepared from thebiological sample. In one embodiment, plasma samples were collected,viral RNA was purified and an RT-PCR methodology was used to amplify apatient derived segment encoding the HIV-1 protease and reversetranscriptase regions. The amplified patient derived segments were thenincorporated, via DNA ligation and bacterial transformation, into anindicator gene viral vector thereby generating a resistance test vector.Resistance test vector DNA was isolated from the bacterial culture andthe phenotypic susceptibility assay was carried out as described inExample 1. The genotype of the protease region was determined by dideoxychain-termination sequencing of the resistance test vector DNA. Sampleswere categorized as having mutations in protease encoding alanine (A),phenylalanine (F), or threonine (T) at position 82, instead of thewild-type valine (V), and the percentage of samples in each categorydisplaying hyper-sensitivity to saquinavir (i.e., fold-change vs.reference of 0.4 or less) was determined. Surprisingly, the percentageof saquinavir hyper-susceptible viruses was much higher amongst virusescontaining V82F than those containing V82A or V82T. This observationimplies that the detection of V82F in protease predicts a positivevirological response to saquinavir treatment.

EXAMPLE 16 Measuring Replication Fitness of Viruses with Mutations inIntegrase

This example provides a means and method for identifying mutations inintegrase that alter replication fitness.

This example provides the means and methods for identifying mutationsthat alter replication fitness for various components of HIV-1replication, including, but not limited to integration, virus assembly,and virus attachment and entry. This example also provides a means andmethod for quantifying the affect that specific mutations in protease,reverse transcriptase, or integrase have on replication fitness. Thismethod can be used for quantifying the effect that specific integrasemutations have on replication fitness and can be used to quantify theeffect of other mutations in other viral genes involved in HIV-1replication, including, but not limited to the gag, pol, and envelopegenes.

Fitness test vectors engineered by site directed mutagenesis to containspecific mutations in integrase were tested in a fitness assay todetermine accurately and quantitatively the relative fitness compared toa well-characterized reference standard.

Genotypic changes that are observed to correlate with resistance tointegrase inhibitors are evaluated by construction of fitness vectorscontaining the specific mutation on a defined, wild-type (drugsusceptible) genetic background. Mutations may be incorporated aloneand/or in combination with other mutations that are thought to modulatethe fitness of a virus. Mutations are introduced into the fitness testvector through any of the widely known methods for site-directedmutagenesis. In one embodiment of this invention the mega-primer PCRmethod for site-directed mutagenesis is used (Sarkar, G. and Sommar, S.S., 1994, Biotechniques 8, 404–407). A fitness test vector containingthe specific mutation or group of mutations are then tested using thefitness assay described in Example 10 and the fitness is compared tothat of a genetically defined wild-type (drug susceptible) fitness testvector which lacks the specific mutations. Observed changes in fitnessare attributed to the specific mutations introduced into the fitnesstest vector. In several related embodiments of the invention, fitnesstest vectors containing site directed mutations in integrase that resultin amino acid substitutions at positions 66, 154, 66 and 153, and 66 and154 are constructed and tested for fitness. As controls, mutants withmultiple changes conferring resistance to reverse transcriptase andprotease inhibitors (MDRC4) and with a mutation in the integrase activesite (D64V) were also tested. The fitness results enable the correlationbetween specific integrase amino acid substitutions and changes in viralfitness.

FIG. 6. Distribution of saquinavir hyper-susceptibility by amino acidchange at position 82.

FIG. 7. Relative luciferase activity of integrase inhibitor-resistantsite-directed mutants.

Table 1: PRI susceptibility of selected patent samples. Virusesdisplaying increased susceptibility to amprenavir (5-fold or greater)were genotyped and found to contain the N88S mutation in PR. Sampleswere listed in order of decreasing susceptibility.

TABLE 1 Sample Prior PRI Fold Change vs. Reference PR Mutations IDExperience SQV IDV RTV NFV AMP PR Mutations 0732 NFV 0.73 2.11 1.72 8.920.08 K14R, I15V, K20T, E35D, M361, R41K, I62V, L63Q, N88S 627 IDV 0.266.16 1.50 21.06 0.09 I13I/V, E35D, M46L, L63P, I64V, I73V, N88S 1208 NFV1.55 3.15 1.22 11.06 0.10 I62V, L63P, V77I, N88S 360 IDV 1.88 6.31 1.4929.95 0.15 I13V, K20M, M36V, N37A, M46I, I62V, L63P, N88S, I93L 0910 NFV1.41 5.17 1.85 16.76 0.16 M46I, L63P, V77I, N88S, I93I/L 3542 IDV 1.287.61 3.36 24.67 0.16 I13V, K14R, N37D, M46, L63P, N88S, I93L 3654 1.807.56 1.95 18.61 0.20 I13V, R41K, M46I, L63P, V77I, N88S, I93L FoldChange Limits: >2.5 < 0.4

TABLE 2 PRI susceptibility of site-directed mutants in PR. Mutationswere introduced into the drug sensitive reference resistance test vectorand the susceptibility to PRIs were determined. Fold Change vs.reference Site-Directed Mutations SQV IDV RTV NFV AMP L63P 1.04 1.121.27 1.43 1.06 L63P, V77I 1.24 1.72 1.73 2.49 0.91 N88S 0.47 1.56 0.362.39 0.04 L63P, N88S 1.44 2.56 0.77 5.20 0.11 L63P, V77I, N88S 1.24 3.091.39 12.89 0.08 M46L, L63P, N88S 1.15 2.30 0.85 6.18 0.12 M46L, L63P,V77I, 1.45 2.97 1.33 12.24 0.14 N88S

Table 3: Relative luciferase activity levels for patient samplevirus-derived resistance test vector pools. The luciferase activity(relative light units, RLU) measured in the absence of drug for thepatient sample was compared to that of the drug sensitive referencecontrol from the same assay run, and expressed as a percentage ofcontrol. These values are from one assay each. All the samples thatcontain the N88S mutations in PR were found to have reduced luciferaseactivity compared to control.

TABLE 3 Relative Luciferase Activity Sample ID PR Mutations (% ofcontrol) 0732 K14R, I15V, K20T, E35D, 8.5 M36I, R41K, I62V, L63Q, N88S627 I131/V, E35D, M46L, L63P, 0.7 I64V, I73V, N88S 1208 I62V, L63P,V77I, N88S 14.2 360 I13V, K20M, M36V, N37A, 2.2 M46I, I62V, L63P, N88S,I93L 0910 M46I, L63P, V77I, N88S, 16.0 I93I/L 3542 I13V, K14R, N37D,M46I, 4.6 L63P, N88S, I93L 3654 I13V, R41K, M46I, L63P, V77I, 12.8 N88S,I93L

Table 4: Relative luciferase activity levels for resistance test vectorscontaining site-directed mutations. The luciferase activity (relativelight units, RLU) measured in the absence of drug for the mutant wascompared to that of the drug sensitive reference control from the sameassay run, and expressed as a percentage of control. These values arefrom one to five assays each, and each value was obtained using anindependent clone for mutants which were tested multiple times. All theconstructs that contain the N88S mutations in PR were found to havereduced luciferase activity compared to control. All the constructs withthe K20T mutation were essentially inactive in the assay.

TABLE 4 Average Luciferase Activity number of Site-Directed Mutations (%of control) clones tested L63P 163.9 1 L63P, V771 75.6 1 N88S 1.0 3L63P, N88S 20.7 2 L63P, V77I, N88S 29.3 2 M46L, L63P, N88S 28.0 2 M46L,L63P, V77I, N88S 53.2 5 K20T, N88S <0.01 5 K20T, L63P, N88S <0.01 1

Table 5: Oligonucleotide primers used for PCR amplification and forgenerating site-directed mutants.

TABLE 5 Primer name: #1: PCR6 5′ CCAATTRYTGTGATATTTCTCATGNTCHTCTTGGG 3′(35-mer) #2: PDS/Apa 5′ CATGTTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTG 3′(42-mer) #3: PDS/Age 5′ CACTCCATGTACCGGTTCTTTTAGAATYTCYCTG 3′ (34-mer)#4: RsrII 5′ ACTTTCGGACCGTCCATTCCTGGCTTTAATTTTACTGGTACAG 3′ (43-mer) #5:K20T 5′ GGGGGGCAATTAACGGAACGTCTATTAG 3′ (28-mer) #6: M46L 5′GATGGAAACCAAAATTGATAGGGGAATTG 3′ (30-mer) #7: L63P 5′GTATGATCAGATACCCATAGAAATCTGC 3′ (28-mer) #8: N88S 5′CTGAGTCAACAGACTTCTTCCAATTATG 3′ (28-mer) R = A or G Y = C or T N = A, C,G, or T H = A, C, or T

TABLE 6 PRI Susceptibility (Fold Change <2.5) of Viruses with Mutationsat 82 and/or 90 Percent of viruses with indicated primary mutation(s)which are drug sensitive (fold change in IC50 < 2.5) V82A/F/S/T and drugV82A/F/S/T L90M L90M RTV 8.0 27.6 3.0 NFV 20.0 8.6 3.0 IDV 22.7 31.0 9.1AMP 53.3 65.5 33.3 SQV 73.3 46.6 21.2

TABLE 7 Correlation Between 82A/F/S/T, Secondary Mutations, and IDVSusceptibility. position n % FC > 2.5 chi square p 24 20 100% <0.005 7127 100% <0.0001 54 38 95% <0.0001 46 35 89% <0.01 10 47 83% <0.05 63 7279% <0.05 82 75 77% all virus with V82A/F/S/T and no other primarymutations.

TABLE 8 Correlation Between 82A/F/S/T, Secondary Mutations, and SQVSusceptibility chi position n % FC > 2.5 square p 20 5 80% <0.001 36 1173% <0.001 24 20 65% <0.0001 71 27 52% <0.0001 54 38 47% <0.0001 10 4740% <0.001 82 75 27% all virus

TABLE 9 Association Between SQV and IDV Susceptibility, V82A/F/S/T, andNumber of Resistance Associated Mutations Number of secondary Number of% with IDV FC % with SQV FC mutations samples > 2.5 > 2.5 1 75 77 27 267 82 30 3 51 88 39 4 38 95 50 5 25 96 60 6 17 100 76 7 5 100 60

TABLE 10 Correlation Between L90M, Secondary Mutations, and IDVSusceptibility. position n % FC > 2.5 chi square p 73 19 89% <0.01 71 1889% <0.001 46 25 88% <0.05 90 58 69% all viruses with L90M and

TABLE 11 Correlation Between L90M, Secondary Mutations, and SQVSusceptibility. position n % FC > 2.5 chi square p 73 19 79% <0.01 71 1878% <0.001 77 25 76% <0.05 10 34 65% <0.05 90 58 55% all viruses

TABLE 12 Association Between SQV and IDV Susceptibility, L90M, andNumber of Resistance Associated Mutations. Number of secondary Number of% w with IDV % w with SQV mutations samples FC > 2.5 FC > 2.5 0 58 69 531 57 70 47 2 56 70 48 3 41 80 68 4 31 87 77 5 14 100 100 6 6 100 100

1. A method for assessing the effectiveness of a candidate HIV antiretroviral compound, said method comprising: a) culturing a host cell in the presence of said candidate HIV antiretroviral compound, wherein said host cell comprises a test vector comprising (i) a nucleic acid segment encoding HIV protease having a mutation at codon 88 and (ii) an indicator gene, wherein expression of said indicator gene depends upon the activity of said HIV protease; b) measuring the amount of expression of said indicator gene in said host cell from step (a); and c) comparing the amount of expression of said indicator gene as measured in step (b) with an amount of expression of said indicator gene measured in said host cell of step (a) cultured in the absence of said candidate HIV antiretroviral compound, wherein the difference between the amount of expression of said indicator gene measured in step (c) relative to that measured in step (b) correlates with the effectiveness of said candidate HIV antiretroviral compound, thereby assessing the effectiveness of said candidate HIV antiretroviral compound.
 2. The method of claim 1, wherein said a nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63 and/or
 77. 3. The method of claim 1, wherein said a nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63, 77 and/or 46, or a combination thereof.
 4. The method of claim 1, wherein said a nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63, 77, 46, 10, 20 and/or 36, or a combination thereof.
 5. A test vector comprising a nucleic acid segment encoding HIV protease having a mutation at codon 88, and an indicator gene, wherein expression of the indicator gene depends upon the activity of said HIV protease.
 6. The test vector of claim 5, wherein said nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63 and/or
 77. 7. The test vector of claim 5, wherein said nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63, 77 and/or 46, or a combination thereof.
 8. The test vector of claim 5, wherein said nucleic acid segment encoding HIV protease further comprises a mutation at codon(s) 63, 77, 46, 10, 20 and/or 36, or a combination thereof. 